Supporting Information Self-Assembly of Cricoid Proteins Induced by ...
Supporting Information Self-Assembly of Cricoid Proteins Induced by “Soft Nanoparticles”: An Approach To Design Multienzyme-Cooperative Antioxidative Systems Hongcheng Sun,† Lu Miao,† Jiaxi Li,† Shuang Fu,† Guo An,† Chengye Si,† Zeyuan Dong,† Quan Luo,† Shuangjiang Yu,‡ Jiayun Xu† and Junqiu Liu*,† †
State Key Laboratory of Supramolecular Structure and Materials, College of
Chemistry, Jilin University, 2699 Qianjin Street, Changchun 130012, China ‡
Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied
Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun 130021, China *
E-mail: [email protected]
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Contents Contents .......................................................................................................................S2 1. Chemicals and Instruments ......................................................................................S3 2. Synthesis of PAMAM Dendrimers ..........................................................................S4 3. Design of Glutathione Peroxidase (GPx) Based on SP1 (SeSP1) .........................S14 4. Design of Superoxide Dismutase (SOD) Based on PD5 (MnPD5) .......................S15 5. Dynamic Light Scattering ......................................................................................S18 6. PD5 Induced Self-Assembly of SP1 (SP1-PD5) ...................................................S20 7. Enzymatic Activity Determination ........................................................................S22 8. The Cellular Uptake Behavior ...............................................................................S22 References ..................................................................................................................S23
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1. Chemicals and Instruments 1.1 Chemicals Dichloromethane, ethyl acetate and acetone were purchased from Beijing Chemical Plant. Methyl acrylate, acryloyl chloride, manganese (II) chloride tetrahydrate, 4-hydroxybenzaldehyde, propanoic acid were purchased from J&K Chemical LTD and used without further purification. Pyrrole was acquired from Fluka (AR grade) and freshly distilled before use. N, N-Dimethylformamide, 1, 2-ethylenediamine and triethylamine were purchased from Sinopharm Chemical Reagent Co. Ltd. and dried over calcium hydride before using. Methanol was obtained from Beijing Chemical Plant and dried with molecular sieves. Xanthine, xanthine oxidase (XOD), reduced nicotinamide adenine dinucleotide phosphate (NADPH), and glutathione disulfide (GSSG) reductase were purchased from Sigma–Aldrich Chemical Co. All restriction enzymes and nucleic acid modifying enzymes were purchased from Sangon Biotech (Shanghai) Co.
1.2 Instruments 1
H NMR spectra was measured on a Bruker 510 spectrometer (500 MHz) using
CDCl3, D2O or DMSO as solvent with tetramethylsilane (TMS) as a reference. Dynamic Light Scattering (DLS) experiments were carried out with Malvern Instrument Zetasizer Nano ZS equipped with a He–Ne laser (633 nm, 4 mW) and an avalanche photodiode detector. Atomic Force Microscopy (AFM) measurements were performed on a NanoScope Multimode AFM (Veeco, USA) using the tapping mode AFM with a SiN4 tip with a radius of 10~20 nm. Transmission Electron Microscopy (TEM) was recorded on a JEM-2100F instrument with an accelerating voltage of 200 kV. Mass spectrometry analyses were performed using Liquid Chromatograph-Mass Spectrometer (LC-MS, Agilent1290-micrOTOF-Q II) or Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS). UV– Vis spectrums were obtained with a Shimadzu 3100 UV-VIS-NIR Recording
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Spectrophotometer interfaced with a personal computer.
2. Synthesis of PAMAM Dendrimers The synthesis of PAMAM dendrimers was shown in Figure S1. 1, 2-Ethylenediamine was employed as core and reacted with methyl acrylate by Michael addition reaction to form quaternary ester. Then, the quaternary ester was aminated with excess 1, 2-ethylenediamine to form quaternary amide, as generation zero (PAMAM G0). We could get different generation PAMAM dendrimer by alternatively Michael additions and amination reactions.1 Specifically, the procedure was as follows:
Figure S1: Synthesis procedures of generation 5 poly(amidoamine) (PAMAM) dendrimer (PD5).
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2.1 Synthesis of Tetramethyl 3, 3', 3'', 3'''-(ethane-1, 2-diylbis (azanetriyl)) tetrapropanoate (PAMAM G-0.5) 1, 2-Ethylenediamine (0.67 mL, 10 mmol) was dissolved in 10 mL of anhydrous methanol. The solution was stirred under nitrogen for 30 min in an ice bath. Then, 10 mL of anhydrous methanol dissolved with methyl acrylate (9.0 mL, 100 mmol) was dropwise added to the solution and kept stirring for 24 h at room temperature. The residual methyl acrylate and solvent was evaporated under reduced pressure to obtain translucent oil. The crude product was purified by silica column chromatography (200–300 mesh) using dichloromethane/ethyl acetate (3:1, v/v) as the eluent. (3.03 g, yield: 75%). 1H-NMR (500 MHz, CDCl3, δ=7.26 ppm) δ=2.43 (t, 8H, -CCH2COO-), 2.48 (s, 4H, -NCH2CH2N-), 2.76 (t, 8H, -NCH2C-), 3.66 (s, 12H, -COOCH3) (Figure S2).
Figure S2: 1H-NMR spectra of PAMAM G-0.5.
2.2 Synthesis of PAMAM G0 1,2-Ethylenediamine (13.3 mL, 200 mmol) was dissolved in 20 mL of anhydrous
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methanol and added dropwise to a solution of PAMAM G -0.5 (2.02 g, 5 mmol) in 20 mL of anhydrous methanol under nitrogen in an ice bath. The mixture was isolated from light and kept stirring for 48 h at room temperature. The reaction mixture was concentrated and repeatedly washed with toluene to remove the residual 1, 2-ethylenediamine (2.50 g, yield: 97%). 1H-NMR (500 MHz, D2O, δ=4.79 ppm) δ=2.45 (t, 8H, -CCH2CONH-), 2.61 (s, 4H, -NCH2CH2N-), 2.72 (t, 8H, -CCH2NH2-), 2.82 (t, 8H, -CH2CCONH-), 3.25 (t, 8H, -CONHCH2-).
2.3 Synthesis of PAMAM G0.5 PAMAM G0 (2.50 g, 4.8 mmol) was dissolved in 30 mL of anhydrous methanol. The solution was stirred under nitrogen for 30 min in an ice bath. Then, 10 mL of anhydrous methanol dissolved with methyl acrylate (17 mL, 192 mmol) was dropwise added to the solution and kept stirring for 24 h at room temperature. The residual methyl acrylate and solvent was evaporated under reduced pressure. The crude product was purified by silica column chromatography (200–300 mesh) using ethyl acetate as the eluent. (4.25 g, yield: 74%). 1H-NMR (500 MHz, CDCl3, δ=7.26 ppm) δ=2.35 (t, 8H, -CCH2CONH-), 2.43 (t, 16H, -CCH2COO-), 2.54 (t, 12H, -NCH2CH2N-, -CONHCCH2N-), 2.76 (t, 24H, -NCH2CCO-), 3.27 (q, 8H, -CONHCH2C-), 3.67 (s, 24H, -COOCH3) (Figure S3).
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Figure S3: 1H-NMR spectra of PAMAM G0.5.
2.4 Synthesis of PAMAM G1 The synthesis of PAMAM G1 was similar to that of PAMAM G0. Specifically, 1, 2-ethylenediamine (16 mL, 240 mmol) was dissolved in 20 mL of anhydrous methanol and added dropwise to a solution of PAMAM G0.5 (2.41 g, 2.0 mmol) in 20 mL of anhydrous methanol under nitrogen in an ice bath. The mixture was kept stirring in dark place for 48 h at room temperature. The reaction mixture was concentrated and repeatedly washed with toluene until the 1,2-ethylenediamine was completely removed. (2.82 g, yield: 99%). 1H-NMR (500 MHz, D2O, δ=4.79 ppm) δ=2.45 (t, 24H, -CCH2CONH-), 2.65 (s, 12H, -NCH2CH2N-), 2.73 (t, 16H, -CCH2NH2-), 2.85 (t, 24H, -CH2CCONH-), 3.26 (16H, -CONHCH2CNH2), 3.32 (8H, -CONHCH2CN-).
2.5 Synthesis of PAMAM G1.5 PAMAM G1.5 was prepared according the synthesis of PAMAM G0.5. Briefly, PAMAM G1 (2.82 g, 2.0 mmol) was dissolved in 30 mL of anhydrous methanol. The
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solution was stirred under nitrogen for 30 min in an ice bath. Then, 20 mL of anhydrous methanol dissolved with methyl acrylate (29 mL, 320 mmol) was dropwise added to the solution and kept stirring for 48 h at room temperature. The residual methyl acrylate and solvent was evaporated under reduced pressure. The crude product was purified by silica column chromatography (200–300 mesh) using ethyl acetate/ methanol (5:1, v/v) as the eluent. (3.5 g, yield: 63%). 1H-NMR (500 MHz, CDCl3, δ=7.26 ppm) δ=2.35 (t, 24H, -CCH2CONH-), 2.42 (t, 32H, -CCH2COO-), 2.52-2.57 (t, 28H, -NCH2CH2N-, -CONHCCH2N-), 2.74 (t, 32H, -NCH2CCOO-), 2.80 (t, 24H, -NCH2CCONH-), 3.26 (t, 24H, -CONHCH2C-), 3.66 (s, 48H, -COOCH3) (Figure S4).
Figure S4: 1H-NMR spectra of PAMAM G1.5.
2.6 Synthesis of PAMAM G2 The synthesis of PAMAM G2 was similar to that of PAMAM G1. 1, 2-ethylenediamine (27 mL, 400 mmol) was dissolved in 20 mL of anhydrous methanol and added dropwise to a solution of PAMAM G1.5 (3.5 g, 1.25 mmol) in 30 mL of anhydrous methanol under nitrogen in an ice bath. The mixture was kept
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stirring in dark place for 48 h at room temperature. Then, the reaction mixture was concentrated and repeatedly washed with toluene to remove the residual 1, 2-Ethylenediamine. (3.9 g, yield: 96%). 1H-NMR (500 MHz, D2O, δ=4.79 ppm) δ=2.44 (56H, -CCH2CONH-), 2.64 (28H, -CCH2N-), 2.73 (32H, -CCH2NH2), 2.83 (56H, -CH2CCONH-), 3.25 (32H, -CONHCH2CNH2), 3.31 (24H, -CONHCH2CN-).
2.7 Synthesis of PAMAM G2.5 PAMAM G2.5 was prepared according the synthesis of PAMAM G1.5. Briefly, PAMAM G2 (2.4 g, 0.74 mmol) was dissolved in 30 mL of anhydrous methanol. The solution was stirred under nitrogen for 30 min in the ice bath. Then, 20 mL of anhydrous methanol dissolved with methyl acrylate (32 mL, 354 mmol) was dropwise added to the solution and kept stirring for 48 h at room temperature. The residual methyl acrylate and solvent was evaporated under reduced pressure. The crude product was purified by silica column chromatography (200–300 mesh) using methanol as the eluent. (3.4 g, yield: 77%). 1H-NMR (500 MHz, CDCl3, δ=7.26 ppm) δ=2.37 (56H, -CCH2CONH-), 2.43 (t, 64H, -CCH2COO-), 2.52-2.57 (m, 60H, -NCH2CH2N-, -CONHCCH2N-), 2.75 (t, 64H, -NCH2CCOO-), 2.80 (m, 56H, -NCH2CCONH-), 3.27 (m, 56H, -CONHCH2C-), 3.66 (s, 96H, -COOCH3) (Figure S5).
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Figure S5: 1H-NMR spectra of PAMAM G2.5.
2.8 Synthesis of PAMAM G3 The synthesis of PAMAM G3 was similar to that of PAMAM G2. Specifically, 1, 2-ethylenediamine (24 mL, 360 mmol) was dissolved in 20 mL of anhydrous methanol and added dropwise to a solution of PAMAM G2.5 (2.7 g, 0.45 mmol) in 40 mL of anhydrous methanol under nitrogen in an ice bath. The mixture was kept stirring in dark place for 72 h at room temperature. The reaction mixture was concentrated and repeatedly washed with toluene. Then, the crude product in aqueous solution was injected into dialysis tube (Spectra/Pro Membrane, MWCO=3,500) for three days. Finally, the solution was freeze-dried to get colorless viscous liquid. (3.0 g, yield: 97%). 1H-NMR (500 MHz, D2O, δ=4.79 ppm) δ=2.43 (120H, -CCH2CONH-), 2.62 (60H, -NCH2CH2N-, -NHCCH2N-), 2.72 (64H, -CCH2NH2), 2.82 (120H, -CH2CCONH-), 3.24 (64H, -CONHCH2CNH2), 3.29 (56H, -CONHCH2CN-).
2.9 Synthesis of PAMAM G3.5 PAMAM G3.5 was prepared according the synthesis of PAMAM G2.5. Briefly,
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PAMAM G3 (3.0 g, 0.435 mmol) was dissolved in 40 mL of anhydrous methanol. The solution was stirred under nitrogen for 30 min at room temperature. 20 mL of anhydrous methanol dissolved with methyl acrylate (50 mL, 557 mmol) was dropwise added to the solution and kept stirring for 48 h at room temperature. The residual methyl acrylate and solvent was evaporated under reduced pressure. The crude product was purified by silica column chromatography (200–300 mesh) using ammonium hydroxide (33 wt. %) /methanol (1/100, v/v) as the eluent. (2.5 g, yield: 46%). 1H-NMR (500 MHz, D2O, δ=4.79 ppm) δ=2.44 (120H, -CCH2CONH-), 2.56 (t, 128H, -CCH2COO-), 2.60-2.66 (m, 124H, -NCH2CH2N-, -CONHCCH2N-), 2.83 (m, 248H, -NCH2CCO-), 3.30 (m, 120H, -CONHCH2C-), 3.70 (s, 192H, -COOCH3) (Figure S6).
Figure S6: 1H-NMR spectra of PAMAM G3.5.
2.10 Synthesis of PAMAM G4 The synthesis of PAMAM G4 was similar to that of PAMAM G3. Specifically, 1, 2-ethylenediamine (26 mL, 388 mmol) was dissolved in 20 mL of anhydrous methanol and added dropwise to a solution of PAMAM G3.5 (2.5 g, 0.202 mmol) in
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40 mL of anhydrous methanol under nitrogen in an ice bath. The mixture was kept stirring in dark place for 72 h at room temperature. The reaction mixture was concentrated and repeatedly washed with toluene. Then, the crude product in aqueous solution was injected into dialysis tube (Spectra/Pro Membrane, MWCO=3,500) for three days. Finally, the solution was freeze-dried to get colorless viscous liquid. (2.8 g, yield: 97%). 1H-NMR (500 MHz, D2O, δ=4.79 ppm) δ=2.43 (248H, -CCH2CONH-), 2.63 (124H, -NCH2CH2N-, -NHCCH2N-), 2.73 (128H, -CCH2NH2), 2.83 (248H, -CH2CCONH-), 3.25 (128H, -CONHCH2CNH2), 3.30 (120H, -CONHCH2CN-).
2.11 Synthesis of PAMAM G4.5 PAMAM G4.5 was prepared according the synthesis of PAMAM G3.5. Briefly, PAMAM G4 (2.8 g, 0.197 mmol) was dissolved in 40 mL of anhydrous methanol. The solution was stirred under nitrogen for 30 min in the ice bath. 30 mL of anhydrous methanol dissolved with methyl acrylate (57 mL, 630 mmol) was dropwise added to the solution and kept stirring for 72 h at room temperature. The residual methyl acrylate and solvent was evaporated under reduced pressure. The crude product was purified by silica column chromatography (200–300 mesh) using ammonium hydroxide (33 wt. %) /methanol (1/20, v/v) as the eluent. (3.3 g, yield: 66%). 1H-NMR (500 MHz, CDCl3, δ=7.26 ppm) δ=2.36 (248H, -CCH2CONH-), 2.43 (t, 256H, -CCH2COO-), 2.53-2.58 (m, 252H, -NCH2CH2N-, -CONHCCH2N-), 2.76 (m, 504H, -NCH2CCO-), 3.26 (m, 248H, -CONHCH2C-), 3.66 (s, 384H, -COOCH3) (Figure S7).
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Figure S7: 1H-NMR spectra of PAMAM G4.5.
2.12 Synthesis of PAMAM G5 (PD5) The synthesis of PD5 was similar to that of PAMAM G4. Specifically, 1, 2-ethylenediamine (34 mL, 503 mmol) was dissolved in 20 mL of anhydrous methanol and added dropwise to a solution of PAMAM G4.5 (3.3 g, 0.131 mmol) in 40 mL of anhydrous methanol under nitrogen in an ice bath. The mixture was kept stirring in dark place for 72 h at room temperature. The reaction mixture was concentrated and repeatedly washed with toluene. Then, the crude product in aqueous solution was injected into dialysis tube (Spectra/Pro Membrane, MWCO=3,500) for three days. Finally, the solution was freeze-dried to get colorless viscous liquid. (3.5 g, yield: 93%). 1H-NMR (500 MHz, D2O, δ=4.79 ppm) δ=2.44 (504H, -CCH2CONH-), 2.63 (252H, -NCH2CH2N-, -NHCCH2N-), 2.73 (256H, -CCH2NH2), 2.83 (504H, -CH2CCONH-), 3.25 (256H, -CONHCH2CNH2), 3.30 (248H, -CONHCH2CN-) (Figure S8).
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Figure S8: 1H-NMR spectra of PD5.
3. Design of Glutathione Peroxidase (GPx) Based on SP1 (SeSP1)
Figure S9: MALDI-TOF mass spectrometry analysis of wild type SP1 (a) and Se-SP1-57Cys (b).
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Figure S10: CD spectra of wild type SP1 (black), SP1-57Cys (green) and Se-SP1-57Cys (red) monitored from 190 to 250 nm.
4. Design of Superoxide Dismutase (SOD) Based on PD5 (MnPD5) 4.1 Synthesis of 5, 10, 15, 20-Tetrakis-(4-hydroxyphenyl)-21, 23HPorphyrin (THPP) 5,10,15,20-Tetrakis-(4-hydroxyphenyl)-21,23H-porphyrin according
to
the
literature
previously
reported
(THPP) (Figure
was
prepared
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Briefly,
4-Hydroxybenzaldehyde (3.66 g, 30 mmol) was dissolved in 250 mL of propanoic acid and the solution was heated to reflux under nitrogen atmosphere. Then, 50 mL of propanoic acid dissolved with fleshly distilled pyrrole (2.01g, 30 mmol) was added dropwise to the stirred solution within 30 min. the mixture was refluxed for 2.5 h under nitrogen atmosphere. When the mixture cooled to room temperature, 200 mL of propanoic acid was evaporated under vacuum and 100 mL of ethanol was added to the solution. Kept at room temperature for 24 h, the resulting precipitate was isolated by vacuum filtration. The filter cake was purified by silica column chromatography (200–300 mesh) using dichloromethane/acetone (5:1, v/v) as the eluent. Yield: 24%.
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1
H-NMR (500 MHz, DMSO-d6): δ=9.94 (s, 4H, OH), 8.86 (s, 8H, J = 9 Hz,
β-pyrrole), 7.99 (d, 8H, J = 8.4 Hz, o-H, hydroxyphenyl), 7.20 (d, 8H, J = 8.4 Hz, m-H, hydroxyphenyl), −2.88 (s, 2H, NH) (Figure S12).
4.2 Metallation of THPP (MnPP) Metallation of THPP was processed as standard procedure (Figure S11). THPP (339 mg, 0.5 mmol) and MnCl2·4H2O (990 mg, 5mmol) were dissolved in DMF (40 mL) and refluxed under nitrogen atmosphere. The reaction was controlled by monitoring the shift of ultraviolet absorption peak from 419 nm to 463 nm. DMF was evaporated under vacuum and the precipitate was purified by silica column chromatography (200–300 mesh) using acetone as the eluent. Yield: 75 %. LC-MS: 731.2 (Figure S13).
Figure S11: Synthesis procedure of THPP and MnPP.
Figure S12: 1H-NMR spectra of THPP
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Figure S13: ESI mass spectrometry of the MnPP.
4.3 Production of MnPD5 The synthesis procedure of MnPD5 was shown in Figure S14. Firstly, acryloyl chloride (14 μL, 0.165 mmol) and TEA (200 μL) were dropwised to DMF (10 mL) dissolved with MnPP (100 mg, 0.136 mmol) in an ice bath. The reaction mixture was kept stirring at room temperature for 12 h to get MnPP-Ac. The residual TEA and DMF were evaporated under vacuum. Then, PD5 (375 mg, 0.013 mmol) was dissolved in 20 mL of anhydrous methanol and added to the samples. The solution was kept stirring at room temperature for 72 h. The solvent was evaporated under vacuum. The crude product was dissolved in deionized water and injected into dialysis tube (Spectra/Pro Membrane, MWCO=3,500) for seven days. Finally, the solution was freeze-dried to get green viscous liquid (MnPD5). The MnPP content of MnPD5 was determined by UV–Vis spectra. Specifically, MnPP was dissolved in methanol at different concentrations (0.005 mM, 0.01mM, 0.02mM and 0.03mM) and the absorption values was determined at 463 nm. The absorption value of MnPP vs concentration to plot and getting the regression equation: y=19.0x+ 0.034. The absorption value of MnPD5 at 3.80 μM was determined to be 0.330, so
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there were 4.1 MnPP molecules per MnPD5 (n=4.1).
Figure S14: Synthesis procedure of MnPD5.
5. Dynamic Light Scattering Dynamic light scattering (DLS) was used to investigate the SP1-PAM5 aggregates using a Malvern Nano_S instrument (Malvern, U.K.) at room temperature. All SP1-PD5 aggregates were analyzed at final SP1 concentration of 1.0 µM in Milli-Q with 0 µM (1:0 of SP1 to PD5), 0.1 µM (1:0.1), 0.2 µM (1:0.2), 0.5 µM (1:0.5) and 1.0 µM (1:1) of PD5, and the size are 8.7 nm, 24 nm, 44 nm, 79 nm and 220 nm, respectively (Figure S9).
Figure S15: The dynamic light scattering (DLS) analysis of the hydrodynamic diameters of pure SP1 (black trace) and the electrostatic induced SP1-PD5 aggregates at the mole ratio of SP1:PD5 in 1:0.1 (red trace), 1:0.2 (green trace), 1:0.5 (blue trace) and 1:1 (magenta trace).
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Figure S16: Stability investigation of the nanowires using the dynamic light scattering (DLS) analysis. (a) The effect of ionic strengths in 0 mM (black), 50 mM (red), 100 mM (green), 200 mM (blue), and 400 mM (purple) NaCl concentration; (b) The effect of pH at pH=2 (black), 6 (red), 9 (green), and 12 (blue); (c) The effect of assembly temperature at 4℃ (black), 25℃ (red), and 37℃ (green); (d) The effect of 20 mM PBS (black), 20 mM HEPES (red), and 20 mM Tris-HCl (green) at pH=7.
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6. PD5 Induced Self-Assembly of SP1 (SP1-PD5)
Figure S17: AFM images of SP1 nanoring (a) and the electrostatic induced SP1-PD5 aggregates at the mole ratio of SP1:PD5 in 1:0.2 (b), 1:0.5 (c) and 1:1 (d) at the final concentration of SP1 in 0.1 µM.
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Figure S18: (a) AFM images of SP1-PD5 nanowires at the final concentration of SP1 in 1.0 µM. The inset chart refers to the height profile along each black line. (b) Bundles of the nanowires in (a). Inset is the electrostatic interaction between amino groups of the PD5 and negative charges (Glu and Asp in red) around the outer surface of the SP1.
Figure S19: DLS dates of the nanowires before (a) and after (b) the catalytic reactions. (c) AFM image of the nanowire structures after catalytic reactions.
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7. Enzymatic Activity Determination
Figure S20: Double-reciprocal plots for the reduction of H2O2 by GSH catalyzed by SeSP1-MnPD5. (a) [E0]/V0 vs. 1/[GSH] (mM−1) at [H2O2] = 0.50 mM. (b) [E0]/V0 vs. 1/[ H2O2] (mM−1) at [GSH] = 1.0 mM. The concentration of SeSP1-MnPD5 was 0.080 μM (SeSP1-MnPD5 as catalytic unit).
8. The Cellular Uptake Behavior
Figure S21: Confocal laser scanning microscopy images of A549 cells after incubation for 3 h with FITC-labeling-PD5 or FITC-labeling-SP1 of the nanowires.
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References 1. Tomalia, D. A.; Baker, H.; Dewald, J.; Hall, M.; Kallos, G.; Martin, S.; Roeck, J.; Ryder, J.; Smith, P. A New Class of Polymers: Starburst-Dendritic Macromolecules. Polym. J. 1985, 17, 117-132. 2. Gao,Y. Z.; Hou, C. X.; Zhou, L. P.; Zhang, D. M.; Zhang, C. Q.; Miao, L.; Wang, Li.; Dong, Z. Y.; Luo Q.; Liu J. Q. A Dual Enzyme Microgel with High Antioxidant Ability Based on Engineered Seleno-Ferritin and Artificial Superoxide Dismutase. Macromol. Biosci. 2013, 13, 808-816.
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State Key Laboratory of Supramolecular Structure and Materials, College of
Chemistry, Jilin University, 2699 Qianjin Street, Changchun 130012, China ‡
Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied
Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun 130021, China *
E-mail: [email protected]
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Contents Contents .......................................................................................................................S2 1. Chemicals and Instruments ......................................................................................S3 2. Synthesis of PAMAM Dendrimers ..........................................................................S4 3. Design of Glutathione Peroxidase (GPx) Based on SP1 (SeSP1) .........................S14 4. Design of Superoxide Dismutase (SOD) Based on PD5 (MnPD5) .......................S15 5. Dynamic Light Scattering ......................................................................................S18 6. PD5 Induced Self-Assembly of SP1 (SP1-PD5) ...................................................S20 7. Enzymatic Activity Determination ........................................................................S22 8. The Cellular Uptake Behavior ...............................................................................S22 References ..................................................................................................................S23
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1. Chemicals and Instruments 1.1 Chemicals Dichloromethane, ethyl acetate and acetone were purchased from Beijing Chemical Plant. Methyl acrylate, acryloyl chloride, manganese (II) chloride tetrahydrate, 4-hydroxybenzaldehyde, propanoic acid were purchased from J&K Chemical LTD and used without further purification. Pyrrole was acquired from Fluka (AR grade) and freshly distilled before use. N, N-Dimethylformamide, 1, 2-ethylenediamine and triethylamine were purchased from Sinopharm Chemical Reagent Co. Ltd. and dried over calcium hydride before using. Methanol was obtained from Beijing Chemical Plant and dried with molecular sieves. Xanthine, xanthine oxidase (XOD), reduced nicotinamide adenine dinucleotide phosphate (NADPH), and glutathione disulfide (GSSG) reductase were purchased from Sigma–Aldrich Chemical Co. All restriction enzymes and nucleic acid modifying enzymes were purchased from Sangon Biotech (Shanghai) Co.
1.2 Instruments 1
H NMR spectra was measured on a Bruker 510 spectrometer (500 MHz) using
CDCl3, D2O or DMSO as solvent with tetramethylsilane (TMS) as a reference. Dynamic Light Scattering (DLS) experiments were carried out with Malvern Instrument Zetasizer Nano ZS equipped with a He–Ne laser (633 nm, 4 mW) and an avalanche photodiode detector. Atomic Force Microscopy (AFM) measurements were performed on a NanoScope Multimode AFM (Veeco, USA) using the tapping mode AFM with a SiN4 tip with a radius of 10~20 nm. Transmission Electron Microscopy (TEM) was recorded on a JEM-2100F instrument with an accelerating voltage of 200 kV. Mass spectrometry analyses were performed using Liquid Chromatograph-Mass Spectrometer (LC-MS, Agilent1290-micrOTOF-Q II) or Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS). UV– Vis spectrums were obtained with a Shimadzu 3100 UV-VIS-NIR Recording
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Spectrophotometer interfaced with a personal computer.
2. Synthesis of PAMAM Dendrimers The synthesis of PAMAM dendrimers was shown in Figure S1. 1, 2-Ethylenediamine was employed as core and reacted with methyl acrylate by Michael addition reaction to form quaternary ester. Then, the quaternary ester was aminated with excess 1, 2-ethylenediamine to form quaternary amide, as generation zero (PAMAM G0). We could get different generation PAMAM dendrimer by alternatively Michael additions and amination reactions.1 Specifically, the procedure was as follows:
Figure S1: Synthesis procedures of generation 5 poly(amidoamine) (PAMAM) dendrimer (PD5).
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2.1 Synthesis of Tetramethyl 3, 3', 3'', 3'''-(ethane-1, 2-diylbis (azanetriyl)) tetrapropanoate (PAMAM G-0.5) 1, 2-Ethylenediamine (0.67 mL, 10 mmol) was dissolved in 10 mL of anhydrous methanol. The solution was stirred under nitrogen for 30 min in an ice bath. Then, 10 mL of anhydrous methanol dissolved with methyl acrylate (9.0 mL, 100 mmol) was dropwise added to the solution and kept stirring for 24 h at room temperature. The residual methyl acrylate and solvent was evaporated under reduced pressure to obtain translucent oil. The crude product was purified by silica column chromatography (200–300 mesh) using dichloromethane/ethyl acetate (3:1, v/v) as the eluent. (3.03 g, yield: 75%). 1H-NMR (500 MHz, CDCl3, δ=7.26 ppm) δ=2.43 (t, 8H, -CCH2COO-), 2.48 (s, 4H, -NCH2CH2N-), 2.76 (t, 8H, -NCH2C-), 3.66 (s, 12H, -COOCH3) (Figure S2).
Figure S2: 1H-NMR spectra of PAMAM G-0.5.
2.2 Synthesis of PAMAM G0 1,2-Ethylenediamine (13.3 mL, 200 mmol) was dissolved in 20 mL of anhydrous
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methanol and added dropwise to a solution of PAMAM G -0.5 (2.02 g, 5 mmol) in 20 mL of anhydrous methanol under nitrogen in an ice bath. The mixture was isolated from light and kept stirring for 48 h at room temperature. The reaction mixture was concentrated and repeatedly washed with toluene to remove the residual 1, 2-ethylenediamine (2.50 g, yield: 97%). 1H-NMR (500 MHz, D2O, δ=4.79 ppm) δ=2.45 (t, 8H, -CCH2CONH-), 2.61 (s, 4H, -NCH2CH2N-), 2.72 (t, 8H, -CCH2NH2-), 2.82 (t, 8H, -CH2CCONH-), 3.25 (t, 8H, -CONHCH2-).
2.3 Synthesis of PAMAM G0.5 PAMAM G0 (2.50 g, 4.8 mmol) was dissolved in 30 mL of anhydrous methanol. The solution was stirred under nitrogen for 30 min in an ice bath. Then, 10 mL of anhydrous methanol dissolved with methyl acrylate (17 mL, 192 mmol) was dropwise added to the solution and kept stirring for 24 h at room temperature. The residual methyl acrylate and solvent was evaporated under reduced pressure. The crude product was purified by silica column chromatography (200–300 mesh) using ethyl acetate as the eluent. (4.25 g, yield: 74%). 1H-NMR (500 MHz, CDCl3, δ=7.26 ppm) δ=2.35 (t, 8H, -CCH2CONH-), 2.43 (t, 16H, -CCH2COO-), 2.54 (t, 12H, -NCH2CH2N-, -CONHCCH2N-), 2.76 (t, 24H, -NCH2CCO-), 3.27 (q, 8H, -CONHCH2C-), 3.67 (s, 24H, -COOCH3) (Figure S3).
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Figure S3: 1H-NMR spectra of PAMAM G0.5.
2.4 Synthesis of PAMAM G1 The synthesis of PAMAM G1 was similar to that of PAMAM G0. Specifically, 1, 2-ethylenediamine (16 mL, 240 mmol) was dissolved in 20 mL of anhydrous methanol and added dropwise to a solution of PAMAM G0.5 (2.41 g, 2.0 mmol) in 20 mL of anhydrous methanol under nitrogen in an ice bath. The mixture was kept stirring in dark place for 48 h at room temperature. The reaction mixture was concentrated and repeatedly washed with toluene until the 1,2-ethylenediamine was completely removed. (2.82 g, yield: 99%). 1H-NMR (500 MHz, D2O, δ=4.79 ppm) δ=2.45 (t, 24H, -CCH2CONH-), 2.65 (s, 12H, -NCH2CH2N-), 2.73 (t, 16H, -CCH2NH2-), 2.85 (t, 24H, -CH2CCONH-), 3.26 (16H, -CONHCH2CNH2), 3.32 (8H, -CONHCH2CN-).
2.5 Synthesis of PAMAM G1.5 PAMAM G1.5 was prepared according the synthesis of PAMAM G0.5. Briefly, PAMAM G1 (2.82 g, 2.0 mmol) was dissolved in 30 mL of anhydrous methanol. The
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solution was stirred under nitrogen for 30 min in an ice bath. Then, 20 mL of anhydrous methanol dissolved with methyl acrylate (29 mL, 320 mmol) was dropwise added to the solution and kept stirring for 48 h at room temperature. The residual methyl acrylate and solvent was evaporated under reduced pressure. The crude product was purified by silica column chromatography (200–300 mesh) using ethyl acetate/ methanol (5:1, v/v) as the eluent. (3.5 g, yield: 63%). 1H-NMR (500 MHz, CDCl3, δ=7.26 ppm) δ=2.35 (t, 24H, -CCH2CONH-), 2.42 (t, 32H, -CCH2COO-), 2.52-2.57 (t, 28H, -NCH2CH2N-, -CONHCCH2N-), 2.74 (t, 32H, -NCH2CCOO-), 2.80 (t, 24H, -NCH2CCONH-), 3.26 (t, 24H, -CONHCH2C-), 3.66 (s, 48H, -COOCH3) (Figure S4).
Figure S4: 1H-NMR spectra of PAMAM G1.5.
2.6 Synthesis of PAMAM G2 The synthesis of PAMAM G2 was similar to that of PAMAM G1. 1, 2-ethylenediamine (27 mL, 400 mmol) was dissolved in 20 mL of anhydrous methanol and added dropwise to a solution of PAMAM G1.5 (3.5 g, 1.25 mmol) in 30 mL of anhydrous methanol under nitrogen in an ice bath. The mixture was kept
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stirring in dark place for 48 h at room temperature. Then, the reaction mixture was concentrated and repeatedly washed with toluene to remove the residual 1, 2-Ethylenediamine. (3.9 g, yield: 96%). 1H-NMR (500 MHz, D2O, δ=4.79 ppm) δ=2.44 (56H, -CCH2CONH-), 2.64 (28H, -CCH2N-), 2.73 (32H, -CCH2NH2), 2.83 (56H, -CH2CCONH-), 3.25 (32H, -CONHCH2CNH2), 3.31 (24H, -CONHCH2CN-).
2.7 Synthesis of PAMAM G2.5 PAMAM G2.5 was prepared according the synthesis of PAMAM G1.5. Briefly, PAMAM G2 (2.4 g, 0.74 mmol) was dissolved in 30 mL of anhydrous methanol. The solution was stirred under nitrogen for 30 min in the ice bath. Then, 20 mL of anhydrous methanol dissolved with methyl acrylate (32 mL, 354 mmol) was dropwise added to the solution and kept stirring for 48 h at room temperature. The residual methyl acrylate and solvent was evaporated under reduced pressure. The crude product was purified by silica column chromatography (200–300 mesh) using methanol as the eluent. (3.4 g, yield: 77%). 1H-NMR (500 MHz, CDCl3, δ=7.26 ppm) δ=2.37 (56H, -CCH2CONH-), 2.43 (t, 64H, -CCH2COO-), 2.52-2.57 (m, 60H, -NCH2CH2N-, -CONHCCH2N-), 2.75 (t, 64H, -NCH2CCOO-), 2.80 (m, 56H, -NCH2CCONH-), 3.27 (m, 56H, -CONHCH2C-), 3.66 (s, 96H, -COOCH3) (Figure S5).
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Figure S5: 1H-NMR spectra of PAMAM G2.5.
2.8 Synthesis of PAMAM G3 The synthesis of PAMAM G3 was similar to that of PAMAM G2. Specifically, 1, 2-ethylenediamine (24 mL, 360 mmol) was dissolved in 20 mL of anhydrous methanol and added dropwise to a solution of PAMAM G2.5 (2.7 g, 0.45 mmol) in 40 mL of anhydrous methanol under nitrogen in an ice bath. The mixture was kept stirring in dark place for 72 h at room temperature. The reaction mixture was concentrated and repeatedly washed with toluene. Then, the crude product in aqueous solution was injected into dialysis tube (Spectra/Pro Membrane, MWCO=3,500) for three days. Finally, the solution was freeze-dried to get colorless viscous liquid. (3.0 g, yield: 97%). 1H-NMR (500 MHz, D2O, δ=4.79 ppm) δ=2.43 (120H, -CCH2CONH-), 2.62 (60H, -NCH2CH2N-, -NHCCH2N-), 2.72 (64H, -CCH2NH2), 2.82 (120H, -CH2CCONH-), 3.24 (64H, -CONHCH2CNH2), 3.29 (56H, -CONHCH2CN-).
2.9 Synthesis of PAMAM G3.5 PAMAM G3.5 was prepared according the synthesis of PAMAM G2.5. Briefly,
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PAMAM G3 (3.0 g, 0.435 mmol) was dissolved in 40 mL of anhydrous methanol. The solution was stirred under nitrogen for 30 min at room temperature. 20 mL of anhydrous methanol dissolved with methyl acrylate (50 mL, 557 mmol) was dropwise added to the solution and kept stirring for 48 h at room temperature. The residual methyl acrylate and solvent was evaporated under reduced pressure. The crude product was purified by silica column chromatography (200–300 mesh) using ammonium hydroxide (33 wt. %) /methanol (1/100, v/v) as the eluent. (2.5 g, yield: 46%). 1H-NMR (500 MHz, D2O, δ=4.79 ppm) δ=2.44 (120H, -CCH2CONH-), 2.56 (t, 128H, -CCH2COO-), 2.60-2.66 (m, 124H, -NCH2CH2N-, -CONHCCH2N-), 2.83 (m, 248H, -NCH2CCO-), 3.30 (m, 120H, -CONHCH2C-), 3.70 (s, 192H, -COOCH3) (Figure S6).
Figure S6: 1H-NMR spectra of PAMAM G3.5.
2.10 Synthesis of PAMAM G4 The synthesis of PAMAM G4 was similar to that of PAMAM G3. Specifically, 1, 2-ethylenediamine (26 mL, 388 mmol) was dissolved in 20 mL of anhydrous methanol and added dropwise to a solution of PAMAM G3.5 (2.5 g, 0.202 mmol) in
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40 mL of anhydrous methanol under nitrogen in an ice bath. The mixture was kept stirring in dark place for 72 h at room temperature. The reaction mixture was concentrated and repeatedly washed with toluene. Then, the crude product in aqueous solution was injected into dialysis tube (Spectra/Pro Membrane, MWCO=3,500) for three days. Finally, the solution was freeze-dried to get colorless viscous liquid. (2.8 g, yield: 97%). 1H-NMR (500 MHz, D2O, δ=4.79 ppm) δ=2.43 (248H, -CCH2CONH-), 2.63 (124H, -NCH2CH2N-, -NHCCH2N-), 2.73 (128H, -CCH2NH2), 2.83 (248H, -CH2CCONH-), 3.25 (128H, -CONHCH2CNH2), 3.30 (120H, -CONHCH2CN-).
2.11 Synthesis of PAMAM G4.5 PAMAM G4.5 was prepared according the synthesis of PAMAM G3.5. Briefly, PAMAM G4 (2.8 g, 0.197 mmol) was dissolved in 40 mL of anhydrous methanol. The solution was stirred under nitrogen for 30 min in the ice bath. 30 mL of anhydrous methanol dissolved with methyl acrylate (57 mL, 630 mmol) was dropwise added to the solution and kept stirring for 72 h at room temperature. The residual methyl acrylate and solvent was evaporated under reduced pressure. The crude product was purified by silica column chromatography (200–300 mesh) using ammonium hydroxide (33 wt. %) /methanol (1/20, v/v) as the eluent. (3.3 g, yield: 66%). 1H-NMR (500 MHz, CDCl3, δ=7.26 ppm) δ=2.36 (248H, -CCH2CONH-), 2.43 (t, 256H, -CCH2COO-), 2.53-2.58 (m, 252H, -NCH2CH2N-, -CONHCCH2N-), 2.76 (m, 504H, -NCH2CCO-), 3.26 (m, 248H, -CONHCH2C-), 3.66 (s, 384H, -COOCH3) (Figure S7).
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Figure S7: 1H-NMR spectra of PAMAM G4.5.
2.12 Synthesis of PAMAM G5 (PD5) The synthesis of PD5 was similar to that of PAMAM G4. Specifically, 1, 2-ethylenediamine (34 mL, 503 mmol) was dissolved in 20 mL of anhydrous methanol and added dropwise to a solution of PAMAM G4.5 (3.3 g, 0.131 mmol) in 40 mL of anhydrous methanol under nitrogen in an ice bath. The mixture was kept stirring in dark place for 72 h at room temperature. The reaction mixture was concentrated and repeatedly washed with toluene. Then, the crude product in aqueous solution was injected into dialysis tube (Spectra/Pro Membrane, MWCO=3,500) for three days. Finally, the solution was freeze-dried to get colorless viscous liquid. (3.5 g, yield: 93%). 1H-NMR (500 MHz, D2O, δ=4.79 ppm) δ=2.44 (504H, -CCH2CONH-), 2.63 (252H, -NCH2CH2N-, -NHCCH2N-), 2.73 (256H, -CCH2NH2), 2.83 (504H, -CH2CCONH-), 3.25 (256H, -CONHCH2CNH2), 3.30 (248H, -CONHCH2CN-) (Figure S8).
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Figure S8: 1H-NMR spectra of PD5.
3. Design of Glutathione Peroxidase (GPx) Based on SP1 (SeSP1)
Figure S9: MALDI-TOF mass spectrometry analysis of wild type SP1 (a) and Se-SP1-57Cys (b).
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Figure S10: CD spectra of wild type SP1 (black), SP1-57Cys (green) and Se-SP1-57Cys (red) monitored from 190 to 250 nm.
4. Design of Superoxide Dismutase (SOD) Based on PD5 (MnPD5) 4.1 Synthesis of 5, 10, 15, 20-Tetrakis-(4-hydroxyphenyl)-21, 23HPorphyrin (THPP) 5,10,15,20-Tetrakis-(4-hydroxyphenyl)-21,23H-porphyrin according
to
the
literature
previously
reported
(THPP) (Figure
was
prepared
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Briefly,
4-Hydroxybenzaldehyde (3.66 g, 30 mmol) was dissolved in 250 mL of propanoic acid and the solution was heated to reflux under nitrogen atmosphere. Then, 50 mL of propanoic acid dissolved with fleshly distilled pyrrole (2.01g, 30 mmol) was added dropwise to the stirred solution within 30 min. the mixture was refluxed for 2.5 h under nitrogen atmosphere. When the mixture cooled to room temperature, 200 mL of propanoic acid was evaporated under vacuum and 100 mL of ethanol was added to the solution. Kept at room temperature for 24 h, the resulting precipitate was isolated by vacuum filtration. The filter cake was purified by silica column chromatography (200–300 mesh) using dichloromethane/acetone (5:1, v/v) as the eluent. Yield: 24%.
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1
H-NMR (500 MHz, DMSO-d6): δ=9.94 (s, 4H, OH), 8.86 (s, 8H, J = 9 Hz,
β-pyrrole), 7.99 (d, 8H, J = 8.4 Hz, o-H, hydroxyphenyl), 7.20 (d, 8H, J = 8.4 Hz, m-H, hydroxyphenyl), −2.88 (s, 2H, NH) (Figure S12).
4.2 Metallation of THPP (MnPP) Metallation of THPP was processed as standard procedure (Figure S11). THPP (339 mg, 0.5 mmol) and MnCl2·4H2O (990 mg, 5mmol) were dissolved in DMF (40 mL) and refluxed under nitrogen atmosphere. The reaction was controlled by monitoring the shift of ultraviolet absorption peak from 419 nm to 463 nm. DMF was evaporated under vacuum and the precipitate was purified by silica column chromatography (200–300 mesh) using acetone as the eluent. Yield: 75 %. LC-MS: 731.2 (Figure S13).
Figure S11: Synthesis procedure of THPP and MnPP.
Figure S12: 1H-NMR spectra of THPP
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Figure S13: ESI mass spectrometry of the MnPP.
4.3 Production of MnPD5 The synthesis procedure of MnPD5 was shown in Figure S14. Firstly, acryloyl chloride (14 μL, 0.165 mmol) and TEA (200 μL) were dropwised to DMF (10 mL) dissolved with MnPP (100 mg, 0.136 mmol) in an ice bath. The reaction mixture was kept stirring at room temperature for 12 h to get MnPP-Ac. The residual TEA and DMF were evaporated under vacuum. Then, PD5 (375 mg, 0.013 mmol) was dissolved in 20 mL of anhydrous methanol and added to the samples. The solution was kept stirring at room temperature for 72 h. The solvent was evaporated under vacuum. The crude product was dissolved in deionized water and injected into dialysis tube (Spectra/Pro Membrane, MWCO=3,500) for seven days. Finally, the solution was freeze-dried to get green viscous liquid (MnPD5). The MnPP content of MnPD5 was determined by UV–Vis spectra. Specifically, MnPP was dissolved in methanol at different concentrations (0.005 mM, 0.01mM, 0.02mM and 0.03mM) and the absorption values was determined at 463 nm. The absorption value of MnPP vs concentration to plot and getting the regression equation: y=19.0x+ 0.034. The absorption value of MnPD5 at 3.80 μM was determined to be 0.330, so
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there were 4.1 MnPP molecules per MnPD5 (n=4.1).
Figure S14: Synthesis procedure of MnPD5.
5. Dynamic Light Scattering Dynamic light scattering (DLS) was used to investigate the SP1-PAM5 aggregates using a Malvern Nano_S instrument (Malvern, U.K.) at room temperature. All SP1-PD5 aggregates were analyzed at final SP1 concentration of 1.0 µM in Milli-Q with 0 µM (1:0 of SP1 to PD5), 0.1 µM (1:0.1), 0.2 µM (1:0.2), 0.5 µM (1:0.5) and 1.0 µM (1:1) of PD5, and the size are 8.7 nm, 24 nm, 44 nm, 79 nm and 220 nm, respectively (Figure S9).
Figure S15: The dynamic light scattering (DLS) analysis of the hydrodynamic diameters of pure SP1 (black trace) and the electrostatic induced SP1-PD5 aggregates at the mole ratio of SP1:PD5 in 1:0.1 (red trace), 1:0.2 (green trace), 1:0.5 (blue trace) and 1:1 (magenta trace).
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Figure S16: Stability investigation of the nanowires using the dynamic light scattering (DLS) analysis. (a) The effect of ionic strengths in 0 mM (black), 50 mM (red), 100 mM (green), 200 mM (blue), and 400 mM (purple) NaCl concentration; (b) The effect of pH at pH=2 (black), 6 (red), 9 (green), and 12 (blue); (c) The effect of assembly temperature at 4℃ (black), 25℃ (red), and 37℃ (green); (d) The effect of 20 mM PBS (black), 20 mM HEPES (red), and 20 mM Tris-HCl (green) at pH=7.
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6. PD5 Induced Self-Assembly of SP1 (SP1-PD5)
Figure S17: AFM images of SP1 nanoring (a) and the electrostatic induced SP1-PD5 aggregates at the mole ratio of SP1:PD5 in 1:0.2 (b), 1:0.5 (c) and 1:1 (d) at the final concentration of SP1 in 0.1 µM.
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Figure S18: (a) AFM images of SP1-PD5 nanowires at the final concentration of SP1 in 1.0 µM. The inset chart refers to the height profile along each black line. (b) Bundles of the nanowires in (a). Inset is the electrostatic interaction between amino groups of the PD5 and negative charges (Glu and Asp in red) around the outer surface of the SP1.
Figure S19: DLS dates of the nanowires before (a) and after (b) the catalytic reactions. (c) AFM image of the nanowire structures after catalytic reactions.
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7. Enzymatic Activity Determination
Figure S20: Double-reciprocal plots for the reduction of H2O2 by GSH catalyzed by SeSP1-MnPD5. (a) [E0]/V0 vs. 1/[GSH] (mM−1) at [H2O2] = 0.50 mM. (b) [E0]/V0 vs. 1/[ H2O2] (mM−1) at [GSH] = 1.0 mM. The concentration of SeSP1-MnPD5 was 0.080 μM (SeSP1-MnPD5 as catalytic unit).
8. The Cellular Uptake Behavior
Figure S21: Confocal laser scanning microscopy images of A549 cells after incubation for 3 h with FITC-labeling-PD5 or FITC-labeling-SP1 of the nanowires.
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References 1. Tomalia, D. A.; Baker, H.; Dewald, J.; Hall, M.; Kallos, G.; Martin, S.; Roeck, J.; Ryder, J.; Smith, P. A New Class of Polymers: Starburst-Dendritic Macromolecules. Polym. J. 1985, 17, 117-132. 2. Gao,Y. Z.; Hou, C. X.; Zhou, L. P.; Zhang, D. M.; Zhang, C. Q.; Miao, L.; Wang, Li.; Dong, Z. Y.; Luo Q.; Liu J. Q. A Dual Enzyme Microgel with High Antioxidant Ability Based on Engineered Seleno-Ferritin and Artificial Superoxide Dismutase. Macromol. Biosci. 2013, 13, 808-816.
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