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Triple stimuli-responsive supramolecular assemblies based on host-guest inclusion complexation between β-cyclodextrin and azobenzene
European Polymer Journal 91 (2017) 396–407
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European Polymer Journal journal homepage: www.elsevier.com/locate/europolj
Macromolecular Nanotechnology
Triple stimuli-responsive supramolecular assemblies based on hostguest inclusion complexation between β-cyclodextrin and azobenzene ⁎
MARK
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Yeqiang Lu, Hui Zou, Hua Yuan, Shuying Gu, Weizhong Yuan , Maoquan Li
Institute of Intervention Vessel, Shanghai 10th People's Hospital, School of Materials Science and Engineering, Tongji University, Shanghai 201804, PR China
AR TI CLE I NF O
AB S T R A CT
Keywords: Stimuli-responsive Supramolecular Inclusion complex Self-assembly Micelles
The host polymer β-cyclodextrin-poly[(2-(2-methoxyethoxy)ethylmethacrylate)-co-oligo(ethylene glycol) methacrylate] [β-CD-P(MEO2MA-co-OEGMA)] was prepared by click chemistry and atom transfer radical polymerization (ATRP), and the guest polymer poly(ε-caprolactone)SS-poly(ethylene glycol) with azobenzene (Azo) group at one end (Azo-PCL-SS-PEG) was synthesized by the combination of ring-opening polymerization (ROP) and esterification reaction. Based on the inclusion complexation between β-CD and Azo groups, the supramolecular polymer β-CD-P(MEO2MA-co-OEGMA)/Azo-PCL-SS-PEG was successfully obtained. Benefitting from the amphiphilicity, the supramolecular polymer could self-assemble into spherical micelles in aqueous solution, and the supramolecular micelles presented obvious UV light-, thermo- and redox-responsive properties. Alternating irradiation of the solution with UV or visible light could induce the reversible supramolecular self-assembly and disassembly of the micelles. When the temperature of the solution increased above the lower critical solution temperature (LCST) of P (MEO2MA-co-OEGMA) chains, the micelles became smaller and aggregated with each other. Moreover, after adding DTT into the micellar solution, the spherical micelles changed into irregular aggregates.
1. Introduction Stimuli-responsive polymers, also known as intelligent and smart polymers, can undergo relatively large and abrupt changes in response to external stimuli, including pH, temperature, redox potential, enzymes and light [1–10]. The self-assembly of stimuliresponsive polymers can generate diverse structures and has wide applications in bioimaging, biotechnology, controlled drug delivery, recyclable catalysis, and so forth [11–19]. In recent years, supramolecular complexes formed by host-guest interactions have been extensively investigated to construct amphiphiles in a simple, dynamic way. Among them, the complexes formed between cyclodextrins (CDs) and azobenzene (Azo) are typical supramolecular assembly systems and have been intensively studied for their unique photo-responsive properties induced by the photochemical trans-cis isomerization of the Azo units [20–24]. It is known that the formation and dissociation of the β-CD/Azo complex can be controlled by light because of the reversible isomerization of Azo under UV and visible light. Ji et al. reported photo-responsive vesicles which could undergo reversible self-assembly and disassembly behaviors via the supramolecular host-guest interaction of β-CD and an amphiphilic Azo-containing block polymer [25]. Li and coworkers synthesized an Azo end-capped pH- and thermo-responsive copolymer (PEG-b-PDMAEMA-Azo), which could construct
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Corresponding authors. E-mail addresses: [email protected] (W. Yuan), [email protected] (M. Li).
http://dx.doi.org/10.1016/j.eurpolymj.2017.04.028 Received 16 March 2017; Received in revised form 15 April 2017; Accepted 20 April 2017 Available online 22 April 2017 0014-3057/ © 2017 Elsevier Ltd. All rights reserved.
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Scheme 1. Synthesis route of the host polymer β-CD-P(MEO2MA-co-OEGMA) and guest polymer Azo-PCL-SS-PEG.
aggregates by supramolecular assembly between β-CD and Azo [26]. Besenbacher and co-workers prepared UV-responsive supramolecular nanofibers from a cyclodextrin-azobenzene inclusion complex through electrospinning [27]. Although a great many stimuli-responsive supramolecular systems have been reported, most of them generally only focused on polymers with single stimulus or dual stimuli because of the inherent difficulty in fabricating multi-stimuli materials from complicated designs and syntheses [28–34]. In contrast, little work has been conducted on the systems with multi-stimuli responsive properties. It is known that multi-stimuli responsive systems can provide a unique opportunity to fine-tune the response to each stimulus independently, and have broad potential applications ranging from drug delivery and tissue engineering to coatings and biological switches [35–40]. Therefore, it is of great importance to design and explore materials with multi-responses. Herein, in this work, a UV light-, thermo- and redox-responsive supramolecular polymer was designed and prepared based on the host-guest inclusion complexation of β-CD and Azo. The host polymer β-cyclodextrin-poly[(2-(2-methoxyethoxy)ethylmethacrylate)co-oligo(ethylene glycol) methacrylate] [β-CD-P(MEO2MA-co-OEGMA)] and the guest copolymer poly(ε-caprolactone)-SS-poly (ethylene glycol) with azobenzene (Azo) group at one end (Azo-PCL-SS-PEG) were firstly synthesized by click chemistry and atom transfer radical polymerization (ATRP) (Scheme 1). The supramolecular polymer was obtained based on the host-guest inclusion complexation between β-CD and Azo. It is known that the host-guest interactions between β-CD and Azo groups present UV lightresponse. Meanwhile, P(MEO2MA-co-OEGMA) is a typical thermo-responsive polymer, which is nontoxic, nonimmunogenic and biocompatible [41]. Moreover, the disulfide bond (SS) is a redox-responsive chemical group which could be broken in the presence of DL-dithiothreitol (DTT) [42]. Therefore, the supramolecular assemblies presented UV light-, thermo- and redox-responsive properties. The conformation and morphology of the supramolecular assemblies could be adjusted by UV irradiation, temperature and redox potential (Scheme 2).
2. Experimental 2.1. Materials ε-Caprolactone (CL, Acros Organic, 99%) was purified with CaH2 by vacuum distillation. β-cyclodextrin (β-CD, Aldrich) was dried 100 °C for 48 h under vacuum after recrystallization from water before use. Tin 2-ethylhexanoate (Sn(Oct)2, Aldrich) was distilled under reduced pressure. Monomethoxy poly(ethylene glycol) (MPEG 5000, Alfa Aesar) was dried by azeotropic distillation with 397
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Scheme 2. The formation, self-assembly, and triple stimuli-responses of the supramolecular polymer.
toluene, and the residual toluene was removed under high vacuum prior to use. N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA; Acros Organic, USA), N,N′-dicyclohexylcarbodiimide (DCC; GL Biochem) and 4-dimethylaminopyridine (DMAP; Fluka), sodium azide (NaN3; Alfa Aesar), 4-(phenyldiazenyl) phenol (Acros Organic, USA), 6-chloro-1-hexanol (Acros Organic, 97%), DLdithiothreitol (DTT, Aldrich, 99%), p-toluenesulfonyl chloride (TsCl, 99%, Acros Organic) and 3,3′-dithiodipropionic acid (DTDP, Aldrich, 99%) were used as received. 2,2′-azobisisobutyronitrile (AIBN; Aldrich, 98%) was recrystallized from ethanol. 2-(2methoxyethoxy)ethyl methacrylate (MEO2MA) and oligo(ethylene glycol) methacrylate (OEGMA, Mn = 475 g/mol) was purchased from Aldrich and passed through a column of activated basic alumina to remove inhibitors. Methylene dichloride (CH2Cl2), tetrahydrofuran (THF), N,N-dimethylformamide (DMF) and triethylamine (Et3N) were dried over CaH2 and distilled under reduced pressure. Propargyl 2-bromoisobutyrate (PBiB) were synthesized according to the methods in the literature [43]. 2.2. Characterization 2.2.1. Nuclear magnetic resonance spectroscopy (NMR) 1 H NMR spectrum was obtained from a Bruker AV 400 NMR spectrometer at ambient temperature with CDCl3, D2O and DMSO-d6 as the solvent. The chemical shifts were relative to tetramethylsilane. 2.2.2. Attenuated total internal reflectance fourier transform infrared (ATR FT-IR) ATR FT-IR spectra of samples were recorded on an Equinoss/Hyperion2000 spectrometer (Bruker, Germany). 2.2.3. Transmission electron microscopy (TEM) The TEM images were obtained using an H-800 (Hitachi, Japan) TEM at an accelerating voltage of 120 kV. The samples for TEM observation were prepared by dropping 10 μL of polymer solutions on copper grids coated with thin films and carbon. 2.2.4. Fluorescence spectroscopy Fluorescence spectra were recorded using an F-2500 spectrometer (Hitachi, Japan) with a xenon lamp source. Fluorescence scans were performed at room temperature in the range of 340–600 nm using increment of 1 nm with the excitation wavelength of 335 nm. The slit widths were set at 5 nm for both the excitation and the emission. 2.2.5. Optical measurement The transmittance of the supramolecular assembly solutions (5.0 mg/mL) at various temperatures was measured at 500 nm on a UV–Vis spectrophotometer (U-3310, Hitachi, Japan) fitted with the temperature controller. Sample cell was thermostated at different temperatures ranging from 25 to 50 °C prior to measurements using the temperature controller. The lower critical solution temperature (LCST) was defined as the temperature producing a 50% decrease in optical transmittance. 2.2.6. UV and visible light irradiation A UV LED irradiator (UVATA, λ0 = 365 nm) and a Vis LED irradiator (CCS, λ0 = 450 nm) were used to induce the photoisomerization of Azo moieties. 2.2.7. Dynamic light scattering (DLS) DLS studies were conducted using a Zetasizer Nano ZS90 instrument (Malvern Instruments) equipped with a multipurpose 398
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autotitrator (MPT-2) at a fixed scattering angle of 90°. The data were processed by cumulants analysis of the experimental correlation function. The hydrodynamic diameters (Dh) were calculated from the computed diffusion coefficient using the Stokes-Einstein equation. 2.3. Synthesis of the host polymer β-CD-P(MEO2MA-co-OEGMA) The synthetic strategy of the host polymer β-CD-P(MEO2MA-co-OEGMA) is illustrated in Scheme 1a. 2.3.1. Synthesis of β-CD-OTs β-CD (50 g, 0.04 mol) was dissolved into 500 mL of 0.4 M sodium hydroxide (8 g, 0.2 mol) solution under stirring, and then 35 g of p-toluenesulfonyl chloride was added in at a very low rate to ensure that the substitution occurred at the C6 position. The mixture was stirred at 5 °C for 30 min. The obtained suspension was filtered and concentrated hydrochloric acid (20 g) was added into the filtrate solution to neutralize the pH value of the solution. The resultant solution was stirred for 1 h. The precipitation was collected by vacuum filtration and washed by water three times. Finally, the product was dried to constant weight in a vacuum oven at room temperature for 48 h (yield: 78%). 1H NMR (DMSO-d6, δ, ppm): 2.45 (s, 3H, -CH3), 3. 3.23–3.38 (m, 14H, H-2,4), 3.60–3.75 (m, 28H, H-3,5,6), 4.79 (d, 2H, H-1), 4,86 (t, 5H, H-1), 5.74 (m, 14H, OH-2,3), 7.45 (d, 2H, H-9), 7.77 (d, 2H, H-8). 2.3.2. Synthesis of Mono-6-deoxy-6-azido-β-cyclodextrin (β-CD-N3) 15.0 g (0.0116 mol) of β-CD-OTs was dissolved in 150 mL water and heated to 80 °C. 4.7 g (0.0723 mol) of sodium azide was added to this solution. After stirring 6 h at 105 °C, the clear solution was precipitated in 900 mL acetone. The white precipitate was isolated by filtering and dried over MgSO4 (yield: 82%). 1H NMR (DMSO-d6, δ, ppm): 3.23–3.38 (m, 14H, H-2,4), 3.65 (m, 28H, H3,5,6), 4.79 (d, 2H, H-1), 4,86 (t, 5H, H-1), 5.74 (m, 14H, OH-2,3). 2.3.3. Synthesis of bromide modified β-CD (β-CD-Br) β-CD-N3 (6.02 g, 5.19 mmol), PBiB (2.07 g, 12.94 mmol), and PMDETA (305 μL, 1.5 mmol) were dissolved in 50 mL dry DMF. After one freeze-pumpthaw cycle, CuBr (0.738 g, 5.27 mmol) was introduced under the protection of argon flow. The reaction flask was degassed by three freeze-pumpthaw cycles and then placed in an oil bath stirring at 40 °C for 48 h. The reaction mixture was then exposed to air, and precipitated in 600 mL ethanol. The precipitation was obtained by suction filtration and washed three times with ethanol, yielding a light blue solid dried overnight in a vacuum oven (yield: 78%). 1H NMR (DMSO-d6, δ, ppm): 1.86 (s, 6H, -COOC(CH3)2Br), 3.34 (br, 14H, H-2,4), 3.65 (br, 28H, H-3,5,6), 4.52 (br, 6H, OH-6), 4.84 (d, 7H, H-1), 5.03 (s, 2H, -CH2-O-), 5.73 (br, 14H, OH-2,3), 8.03–8.15 (d, 2H, -CH- of the 1, 2, 3-triazole group). 2.3.4. Synthesis of the host polymer β-CD-P(MEO2MA-co-OEGMA) β-CD-P(MEO2MA-co-OEGMA) was synthesized by ATRP of MEO2MA monomer and OEGMA monomer with β-CD-Br as the initiator and CuBr/PMDETA as the catalyst system. A dried Schlenk flask with a magnetic stirrer was charged with β-CD-Br (0.6 g, 0.44 mmol), MEO2MA (7.38 g, 39.25 mmol), OEGMA (1.62 g, 3.407 mmol), CuBr (63.07 mg, 0.44 mmol) and anhydrous DMF (6 mL). The flask was degassed with three freeze-evacuate-thaw cycles. PMDETA (92.86 μL, 0.44 mmol) was deoxygenated by bubbling dry argon before injection into the reaction system by syringe. The polymerization reaction was performed at 80 °C for 4 h. After being cooled to room temperature, the reaction flask was opened to air, and the crude product was diluted with THF and passed through a neutral oxide alumina column to remove the copper catalysts. Then the filtered solution was purified by dialysis (molecular weight cut-off: 3000 Da) against water to remove the catalyst and unreacted monomers. After lyophilization to remove the water, the final purified product was obtained (yield: 83%). Mn, GPC = 12,680 g/mol, Mw/Mn = 1.26. Mn, NMR = 14,000 g/mol. 1H NMR (DMSO-d6, δ, ppm): 0.58–1.24 (m, 3H, CH2C(CH3)), 1.59–2.21 (m, 2H, CH2C(CH3)), 3.39 (s, 3H, OCH2CH2OCH3), 3.48–3.79 (t, 4H, OCH2CH2O), 4.09 (t, 2H, COOCH2CH2O), 5.03 (s, 2H, -CH2-O-), 5.73 (m, 14H, OH-2,3), 8.13 (s, 1H, -CH- of the 1, 2, 3-triazole group). 2.4. Synthesis of the guest polymer Azo-PCL-SS-PEG The synthetic strategy of the guest polymer Azo-PCL-SS-PEG is illustrated in Scheme 1b. 2.4.1. Synthesis of Azo-OH The 6-(4-(phenyldiazenyl) phenoxy) hexan-1-ol (Azo-OH) was prepared using the similar procedures according to the literature [44]. 4-(phenyldiazenyl) phenol (4.95 g, 25 mmol), 6-chloro-1-hexanol (5.1 g, 37.5 mmol), potassium carbonate (5.175 g, 37.5 mmol) and a trace amount of potassium iodide were dissolved in 100 mL DMF. The mixture solution was refluxed for 12 h and washed with a large amount of water. The product was extracted with chloroform, dried by anhydrous MgSO4. After the solvent was removed by evaporation, the crude product was purified by recrystallization from ethanol to obtain the purified product (yield: 35%). 1H NMR (CDCl3, δ, ppm): 1.44–1.54 (m, 4H, -CH2-CH2-CH2-CH2-OH), 1.63(m, 2H, -CH2-CH2-OH), 1.84(m, 2H, -Ph-O-CH2CH2-), 3.67 (t, 2H, -CH2-OH), 4.05 (t, 2H, -Ph-O-CH2-), 7.00–7.86 (m, 9H, -CH- of the azobenzene group). 2.4.2. Synthesis of Azo-PCL-OH Azo-PCL-OH was synthesized by ROP of CL with Azo-OH as the initiator. Briefly, CL (13.389 g, 0.1175 mol), Azo-OH (1 g, 399
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3.34 mmol), and a catalytic amount of Sn(Oct)2 (140 mmol) were added to a fire-dried polymerization tube quickly. The tube was then connected to a Schlenk line, where exhausting-refilling processes were repeated thrice. The tube was put into an oil bath at 120 °C under argon atmosphere with stirring and cooled to room temperature after polymerization for 24 h. The resulting product was dissolved in chloroform and precipitated twice in methanol. The purified polymer was dried in vacuum at room temperature (yield: 95%). Mn, NMR = 4860 g/mol. 1H NMR (CDCl3, δ, ppm): 1.40 (m, 2H, CH2CH2CH2CH2CH2O), 1.65 (m, 4H, CH2CH2CH2CH2CH2O), 2.30 (t, 2H, CH2CH2CH2CH2CH2O), 4.05 (t, 2H, CH2CH2CH2CH2CH2O), 7.00–7.86 (m, 9H, -CH- of the azobenzene group). 2.4.3. Synthesis of Azo-PCL-SS-COOH Azo-PCL-OH (5 g, 1.166 mmol), DTDP (0.245 g, 11.7 mmol), DCC (0.72 g, 3.5 mmol), DMAP (0.2134 g, 1.75 mmol) and THF (25 mL) were added into a 50 mL around-bottomed flask. The reaction was carried out at room temperature for 36 h under argon atmosphere with stirring. The reaction byproduct dicyclohexylcarbodiurea (DCU) was removed by vacuum filtration. The solution was concentrated by rotary evaporator and precipitated in methanol. The product was dried to constant weight in a vacuum oven at room temperature for 48 h (yield: 91%). Mn, NMR = 5050 g/mol. 1H NMR (CDCl3, δ, ppm):1.40 (m, 2H, CH2CH2CH2CH2CH2O), 1.65 (m, 4H, CH2CH2CH2CH2CH2O), 2.30 (t, 2H, CH2CH2CH2CH2CH2O), 2.9 (t, 2H, SSCH2CH2COOH), 3.0 (t, 2H, SSCH2CH2OC(O)), 4.05 (t, 2H, CH2CH2CH2CH2CH2O), 7.00–7.86 (m, 9H, -CH- of the azobenzene group). 2.4.4. Synthesis of Azo-PCL-SS-PEG Azo-PCL-SS-COOH (2 g, 4.44 mmol), MPEG (2.2 g, 4.44 mmol), DMAP (0.195 g, 6.66 mmol) and DCC (0.275 g, 13.32 mmol) were dissolved in 25 mL CH2Cl2. And the reaction was carried out at room temperature for 48 h under vigorous stirring. The reaction byproduct dicyclohexylcarbodiurea (DCU) was removed by vacuum filtration. After the CH2Cl2 solvent was removed by evaporation, the crude product was then dialyzed against distilled water for 72 h using a dialysis bag (molecular weight cut-off: 8000–14,000 Da) to remove unreacted stuffs. After lyophilization to remove the water, the final purified product was obtained (yield: 85%). Mn, 1 GPC = 10,320 g/mol, Mw/Mn = 1.24. Mn, NMR = 11,100 g/mol. H NMR (CDCl3, δ, ppm):1.40 (m, 2H, CH2CH2CH2CH2CH2O), 1.65 (m, 4H, CH2CH2CH2CH2CH2O), 2.30 (t, 2H, CH2CH2CH2CH2CH2O), 3.65 (t, 4H, OCH2CH2O), 4.05 (t, 2H, CH2CH2CH2CH2CH2O), 7.00–7.86 (m, 9H, -CH- of the azobenzene group). 2.5. Preparation of the supramolecular assemblies The Azo-PCL-SS-PEG (22.2 mg) and β-CD-P(MEO2MA-co-OEGMA) (28 mg) were dissolved in DMF (10 mL). It was then mixed manually followed by sonication for 30 min. Then the mixed solution was dialyzed against distilled water for 72 h using a dialysis bag (molecular weight cut-off: 8000–14,000 Da) to obtain the supramolecular assemblies. 2.6. Critical micelle concentration (CMC) measurement of the supramolecular assemblies in aqueous solutions The critical micelle concentration (CMC) was measured using pyrene as a fluorescence probe. 5 mg pyrene was dissolved in 10 mL acetone and then 10 μL of the solution was added into each cuvette. The acetone was allowed to evaporate for 2 h at room temperature. Then 2.0 mL of aqueous supramolecular assembly solution ranging from 1.95 mg/L to 1000 mg/L were added into the pyrene-containing cuvette separately. Upon sonication for 10 min, the solutions were kept at room temperature and equilibrated for 24 h before fluorescent emission measurements with the excitation wavelength of 335 nm. The spectra were recorded in the 340–600 nm wavelength range. For each spectrum obtained, the intensity ratio of the first and third peaks, I1/I3, was calculated. The CMC was estimated as the concentration at which I1/I3 began to drop, suggesting that the micellation of the polymer occurred. 3. Results and discussion 3.1. Synthesis of the host polymer β-CD-P(MEO2MA-co-OEGMA) The products of β-CD-OTs and β-CD-N3 were characterized by 1H NMR (Fig. 1a and b) and FT-IR (Fig. 2b and c). As shown in Fig. 1a and b, all the protons signals assigned to β-CD-OTs and β-CD-N3 could be clearly observed, indicating the successful preparation of the pure β-CD-OTs and β-CD-N3. Moreover, a comparison of the FT-IR spectra of β-CD-OTs and β-CD-N3 in Fig. 2b and c revealed the appearance of a strong absorbance peak at 2100 cm−1 for β-CD-N3, which was characteristic absorption peak of the azide group, further confirming the successful synthesis of β-CD-N3. β-CD-Br was obtained via the click reaction of β-CDN3 and excess PBiB. As shown in the Fig. 1c, the peaks at about 8.1 of methine proton in 1,2,3-triazole ring (peak a) proved the formation of the functional initiator β-CD-Br. As shown in the Fig. 2d, after the click reaction, the absorption band at 2100 cm−1 disappeared, and the peck of carbonyl group at 1730 cm−1 occurred, indicating the occurrence of click reaction of β-CD-N3 and PBiB. β-CD-P(MEO2MA-co-OEGMA) was synthesized by ATRP of MEO2MA and OEGMA with β-CD-Br as initiator and CuBr/PMDETA as catalyst system. The feed ratio of MEO2MA and OEGMA was 92:8. The peaks of protons from MEO2MA and OEGMA were corresponded to the peaks of f and g in Fig. 1d. All characteristic signals of the host polymer β-CD-P(MEO2MA-co-OEGMA) could be observed and the corresponding peaks have been assigned. And the number-average molecular weight calculated by 1H NMR (Mn, NMR) was 14,000 g/mol. 400
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Fig. 1. 1H NMR spectrum of (a) β-CD-OTs, (b) β-CD-N3, (c) β-CD-Br, and (d) β-CD-P(MEO2MA-co-OEGMA).
Fig. 2. FT-IR spectra of (a) β-CD, (b) β-CD-OTs, (c) β-CD-N3 and (d) β-CD-Br.
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Fig. 3. 1H NMR spectrum of (a) Azo-OH, (b) Azo-PCL-SS-COOH, (c) Azo-PCL-OH, and (d) Azo-PCL-SS-PEG.
3.2. Synthesis of the guest polymer Azo-PCL-SS-PEG Azo-OH was synthesized by the Williamson etherification of 4-(phenyldiazenyl) phenol and 6-chloro-1-hexanol. Fig. 3a shows the H NMR spectrum of Azo-OH. All the protons of Azo-OH could be observed. The integral area ratio of peak e (4.08 ppm) to peak c (7.95 ppm) was 1: 2, indicating the successful synthesis of Azo-OH. Azo-PCL-OH was synthesized by ROP of CL with the obtained Azo-OH as the initiator. The 1H NMR spectrum of Azo-PCL-OH is shown in Fig. 3b. All the proton signals of the polymer could be clearly detected. The degree of polymerization of CL was obtained from the integration of the proton signals at 4.05 ppm (peak m) to that at 3.65 ppm (peak m’), which was about 35. Azo-PCL-SS-COOH was synthesized by DCC reaction. The 1H NMR spectrum of AzoPCL-SS-COOH is shown in Fig. 3c. The peaks of protons from Azo and PCL segments can be clearly observed. According to the proton peaks of p and q, it can be found that the disulfide bond of 3,3′-dithiodipropionic acid was successfully attached to the polymer. The Azo-PCL-SS-PEG copolymer was prepared through the esterification reaction of Azo-PCL-SS-COOH with MPEG. From the 1H NMR spectrum of Azo-PCL-SS-PEG (Fig. 3d), the proton peak i (3.6 ppm) and peak j (3.4 ppm) could be well assigned to PEG. And the number-average molecular weight calculated by 1H NMR (Mn, NMR) was 11,100 g/mol. The GPC traces of β-CD-P(MEO2MA-co-OEGMA) and Azo-PCL-SS-PEG are shown in Fig. 4. The traces were mono-modal, indicating that the pure polymers were obtained. The number-average molecular weights (Mn, GPC) of β-CD-P(MEO2MA-co-OEGMA) and Azo-PCL-SS-PEG were 12,680 g/mol and 10,320 g/mol, respectively. 1
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Fig. 4. GPC traces of β-CD-P(MEO2MA-co-OEGMA) and Azo-PCL-SS-PEG.
3.3. Self-assembly of the supramolecular polymer Resulting from the amphiphilic property, the supramolecular polymer could self-assemble into spherical micelles in aqueous solutions. Fig. 5a shows the TEM images of the supramolecular assemblies. It can be seen that the supramolecular assemblies had a regular spherical morphology with a diameter of ∼85 nm, indicating that they were micelles. Consistently, the hydrodynamic diameter (Dh) of the supramolecular assemblies determined using DLS (Fig. 5b) was 109.85 nm, and the particle dispersion index (PDI) was 0.313. The CMC of the supramolecular polymer in aqueous solutions was measured using pyrene as the fluorescent probe, and the CMC of the supramolecular polymer was 81.8 mg/L as shown in Fig. 5c. 3.4. UV light -responsive behaviors of the supramolecular assemblies Due to the UV-response of the β-CD/Azo inclusion complex, the supramolecular assemblies demonstrated UV-responsive properties in aqueous solution. Fig. 6a shows the TEM images of the supramolecular assemblies after UV light irradiation. The size of the supramolecular assemblies decreased from ∼85 nm to ∼45 nm after UV irradiation, and the Dh value changed from 109.85 nm
Fig. 5. (a) TEM image and (b) DLS data of the supramolecular assemblies at 25 °C (Concentration: 0.5 mg/mL), (c) CMC measurement of the supramolecular polymer.
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Fig. 6. (a) TEM image and (b) DLS data of the supramolecular assemblies after UV irradiation at 365 nm for 1 min (Concentration: 0.5 mg/mL).
to 51.75 nm with a PDI of 0.526 (Fig. 6b). This was because the trans-Azo changed to cis-state upon UV light irradiation, and then departed from the cavity of β-CD. As a result, the hydrophilic-hydrophobic balance of the system altered, leading to the decrease of the size of spherical micelles. The light-response of the supramolecular assemblies was further traced and confirmed by UV–vis spectra. The absorption bands at 355 and 440 nm are ascribed to the π-π∗ transition of the trans-Azo form and n-π∗ transition of the cis-Azo form of the Azo group, respectively. As shown in Fig. 7a, upon irradiation by UV light, the absorption peak at 355 nm was remarkably decreased, while the absorption at 440 nm was slightly increased, indicating the photo-isomerization of the trans-Azo to cis-Azo. When the same sample was irradiated with visible light, the peak at 355 nm increased remarkably, and the peak at around 440 nm decreased concomitantly, which indicated the reversible isomerization of Azo groups from cis to trans state (Fig. 7b). Furthermore, on alternating irradiation of the sample with UV and visible light, the reversible trans to cis photo-isomerization process could be recycled many times (Fig. 7c). Fig. 7d shows the TEM images of the supramolecular assemblies after four UV–visible light irradiation cycles. The supramolecular assemblies returned to the initial state with the size of ∼90 nm. The Dh value of the supramolecular assemblies was 112.26 nm with a PDI was 0.313 (Fig. 7e).
Fig. 7. UV–Vis spectrum of supramolecular assemblies: (a) 365 nm UV irradiation induced trans-to-cis transition, (b) 450 nm visible light irradiation induced cis-totrans transition, (c) changes of Azo-CD inclusion upon alternating irradiation with 365 nm UV light and 450 nm visible light, (d) TEM image and (e) DLS data of the supramolecular assemblies after four UV–visible light irradiation cycles at 25 °C (Concentration: 0.5 mg/mL).
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Fig. 8. (a) Optical transmittance and (b) temperature dependence of hydrodynamic diameter (Dh) of the supramolecular assemblies, (c) TEM image and (d) DLS data of the supramolecular assemblies after heating to 50 °C (Concentration: 0.5 mg/mL).
3.5. Thermo-responsive behaviors of the supramolecular assemblies Benefitting from the thermo-response of P(MEO2MA-co-OEGMA) segments, the supramolecular assemblies presented thermoresponsive properties. Fig. 8a shows the transmittance curve of the supramolecular assembly solution. It can be seen that the transmittance curve showed a sharp decreasing transition during heating from 38 °C to 40 °C and the lower critical solution temperature (LCST) value was calculated to be ∼38.8 °C. The Dh values of the supramolecular assemblies in aqueous solution as a function of temperature are shown in Fig. 8b. At relatively lower temperatures, the Dh values were small and changed little. In contrast, when at higher temperature ranges (> 37 °C), the Dh values increased significantly with the rise of temperature, which was due to the aggregation among micelles. The reason for the thermo-response of the supramolecular assemblies in aqueous solutions was that P(MEO2MA-co-OEGMA) chains existed in random coil conformations at low temperature range due to the hydrogen-bonding reaction between the ether oxygen and water molecules. When the temperature increased to a critical value (> LCST), P(MEO2MAco-OEGMA) chains shrank into a globular structure and became hydrophobic because the hydrogen bonds between the ether oxygen of P(MEO2MA-co-OEGMA) and water molecules collapsed. As a result, the intermolecular hydrophobic attractions were thermodynamically favoured and the supramolecular assemblies tended to agglomerate, leading to the increase in Dh and visible turbidity. Fig. 8c shows the TEM image of supramolecular assemblies after heating to the temperature of 50 °C. It can be observed that the spherical micelles became smaller and aggregated with each other. At the same time, the DLS trace of the heated solution was asymmetric with a shoulder and the PDI value was 0.813 as shown in Fig. 8d, indicating that the aggregates were irregular. The thermo-response of the supramolecular assemblies was further confirmed using 1H NMR spectra in D2O. As shown in Fig. 9, the chemical shifts in P(MEO2MA-co-OEGMA) could be observed and their intensities decreased with increasing temperature. This decrease was because that P(MEO2MA-co-OEGMA) segments underwent a configuration transition from a random coil conformation to a globular structure and the P(MEO2MA-co-OEGMA) solubility decreased after the collapse of the hydrogen-bonding interaction between the ether oxygen and water molecules upon heating. It can be seen that peak d was remained in the spectra. This was because at high temperatures the water-soluble PEG segments of the guest polymer were stretched out in the solution to stabilize the micelles despite the P(MEO2MA-co-OEGMA) segments were hydrophobic.
3.6. Redox-responsive behaviors of the supramolecular assemblies Due to the existence of the disulfide bonds, the supramolecular assemblies presented redox responses. Fig. 10a shows the TEM image of assemblies treated with 10 mM DTT over 24 h. It can be seen that the regular spherical micelles changed into irregular 405
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Fig. 9. 1H NMR spectra of the supramolecular polymer in D2O conducted at different temperatures.
aggregates with different sizes, and the Dh of assemblies increased to 215.06 nm with a wide PDI of 0.743 after adding DTT (Fig. 10b). The reason for this change was that the disulfide bonds linking the PCL and PEG blocks were broken in the reaction with DTT. As the reductive cleavage of the disulfide bonds occurred, the PEG segments would shed. Then the hydrophilic-hydrophobic balance of supramolecular assemblies was broken and the micelles became unstable, and then the irregular aggregates were formed.
4. Conclusions In summary, novel UV light-, thermo- and redox-responsive supramolecular micelles consisted of β-CD-P(MEO2MA-co-OEGMA)/ Azo-PCL-SS-PEG have been successfully prepared based on the inclusion complexation between β-CD and Azo. The conformation and morphology of the supramolecular micelles could be adjusted by UV irradiation, temperature and redox potential. Alternating irradiation of the solution with UV or visible light could induce the reversible supramolecular self-assembly and disassembly of micelles. When the temperatures were higher than a critical value, the micelles became smaller and aggregated with each other. After adding DTT into the micellar system, the spherical micelles changed into irregular and became smaller. Therefore, the size and morphology of the supramolecular assemblies could be fine-tuned to each stimulus independently. As a result, the supramolecular assemblies with controllable size and morphology may have potential applications in nanotechnology and smart-material fields.
Acknowledgment The authors gratefully acknowledge the financial support of the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry and the National Key Technology R & D Program (no. 2012BAI15B06).
Fig. 10. (a) TEM image, and (b) DLS data of the supramolecular assemblies after addition of DTT (10 mM) over 24 h (Concentration: 0.5 mg/mL).
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European Polymer Journal journal homepage: www.elsevier.com/locate/europolj
Macromolecular Nanotechnology
Triple stimuli-responsive supramolecular assemblies based on hostguest inclusion complexation between β-cyclodextrin and azobenzene ⁎
MARK
⁎
Yeqiang Lu, Hui Zou, Hua Yuan, Shuying Gu, Weizhong Yuan , Maoquan Li
Institute of Intervention Vessel, Shanghai 10th People's Hospital, School of Materials Science and Engineering, Tongji University, Shanghai 201804, PR China
AR TI CLE I NF O
AB S T R A CT
Keywords: Stimuli-responsive Supramolecular Inclusion complex Self-assembly Micelles
The host polymer β-cyclodextrin-poly[(2-(2-methoxyethoxy)ethylmethacrylate)-co-oligo(ethylene glycol) methacrylate] [β-CD-P(MEO2MA-co-OEGMA)] was prepared by click chemistry and atom transfer radical polymerization (ATRP), and the guest polymer poly(ε-caprolactone)SS-poly(ethylene glycol) with azobenzene (Azo) group at one end (Azo-PCL-SS-PEG) was synthesized by the combination of ring-opening polymerization (ROP) and esterification reaction. Based on the inclusion complexation between β-CD and Azo groups, the supramolecular polymer β-CD-P(MEO2MA-co-OEGMA)/Azo-PCL-SS-PEG was successfully obtained. Benefitting from the amphiphilicity, the supramolecular polymer could self-assemble into spherical micelles in aqueous solution, and the supramolecular micelles presented obvious UV light-, thermo- and redox-responsive properties. Alternating irradiation of the solution with UV or visible light could induce the reversible supramolecular self-assembly and disassembly of the micelles. When the temperature of the solution increased above the lower critical solution temperature (LCST) of P (MEO2MA-co-OEGMA) chains, the micelles became smaller and aggregated with each other. Moreover, after adding DTT into the micellar solution, the spherical micelles changed into irregular aggregates.
1. Introduction Stimuli-responsive polymers, also known as intelligent and smart polymers, can undergo relatively large and abrupt changes in response to external stimuli, including pH, temperature, redox potential, enzymes and light [1–10]. The self-assembly of stimuliresponsive polymers can generate diverse structures and has wide applications in bioimaging, biotechnology, controlled drug delivery, recyclable catalysis, and so forth [11–19]. In recent years, supramolecular complexes formed by host-guest interactions have been extensively investigated to construct amphiphiles in a simple, dynamic way. Among them, the complexes formed between cyclodextrins (CDs) and azobenzene (Azo) are typical supramolecular assembly systems and have been intensively studied for their unique photo-responsive properties induced by the photochemical trans-cis isomerization of the Azo units [20–24]. It is known that the formation and dissociation of the β-CD/Azo complex can be controlled by light because of the reversible isomerization of Azo under UV and visible light. Ji et al. reported photo-responsive vesicles which could undergo reversible self-assembly and disassembly behaviors via the supramolecular host-guest interaction of β-CD and an amphiphilic Azo-containing block polymer [25]. Li and coworkers synthesized an Azo end-capped pH- and thermo-responsive copolymer (PEG-b-PDMAEMA-Azo), which could construct
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Corresponding authors. E-mail addresses: [email protected] (W. Yuan), [email protected] (M. Li).
http://dx.doi.org/10.1016/j.eurpolymj.2017.04.028 Received 16 March 2017; Received in revised form 15 April 2017; Accepted 20 April 2017 Available online 22 April 2017 0014-3057/ © 2017 Elsevier Ltd. All rights reserved.
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Scheme 1. Synthesis route of the host polymer β-CD-P(MEO2MA-co-OEGMA) and guest polymer Azo-PCL-SS-PEG.
aggregates by supramolecular assembly between β-CD and Azo [26]. Besenbacher and co-workers prepared UV-responsive supramolecular nanofibers from a cyclodextrin-azobenzene inclusion complex through electrospinning [27]. Although a great many stimuli-responsive supramolecular systems have been reported, most of them generally only focused on polymers with single stimulus or dual stimuli because of the inherent difficulty in fabricating multi-stimuli materials from complicated designs and syntheses [28–34]. In contrast, little work has been conducted on the systems with multi-stimuli responsive properties. It is known that multi-stimuli responsive systems can provide a unique opportunity to fine-tune the response to each stimulus independently, and have broad potential applications ranging from drug delivery and tissue engineering to coatings and biological switches [35–40]. Therefore, it is of great importance to design and explore materials with multi-responses. Herein, in this work, a UV light-, thermo- and redox-responsive supramolecular polymer was designed and prepared based on the host-guest inclusion complexation of β-CD and Azo. The host polymer β-cyclodextrin-poly[(2-(2-methoxyethoxy)ethylmethacrylate)co-oligo(ethylene glycol) methacrylate] [β-CD-P(MEO2MA-co-OEGMA)] and the guest copolymer poly(ε-caprolactone)-SS-poly (ethylene glycol) with azobenzene (Azo) group at one end (Azo-PCL-SS-PEG) were firstly synthesized by click chemistry and atom transfer radical polymerization (ATRP) (Scheme 1). The supramolecular polymer was obtained based on the host-guest inclusion complexation between β-CD and Azo. It is known that the host-guest interactions between β-CD and Azo groups present UV lightresponse. Meanwhile, P(MEO2MA-co-OEGMA) is a typical thermo-responsive polymer, which is nontoxic, nonimmunogenic and biocompatible [41]. Moreover, the disulfide bond (SS) is a redox-responsive chemical group which could be broken in the presence of DL-dithiothreitol (DTT) [42]. Therefore, the supramolecular assemblies presented UV light-, thermo- and redox-responsive properties. The conformation and morphology of the supramolecular assemblies could be adjusted by UV irradiation, temperature and redox potential (Scheme 2).
2. Experimental 2.1. Materials ε-Caprolactone (CL, Acros Organic, 99%) was purified with CaH2 by vacuum distillation. β-cyclodextrin (β-CD, Aldrich) was dried 100 °C for 48 h under vacuum after recrystallization from water before use. Tin 2-ethylhexanoate (Sn(Oct)2, Aldrich) was distilled under reduced pressure. Monomethoxy poly(ethylene glycol) (MPEG 5000, Alfa Aesar) was dried by azeotropic distillation with 397
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Scheme 2. The formation, self-assembly, and triple stimuli-responses of the supramolecular polymer.
toluene, and the residual toluene was removed under high vacuum prior to use. N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA; Acros Organic, USA), N,N′-dicyclohexylcarbodiimide (DCC; GL Biochem) and 4-dimethylaminopyridine (DMAP; Fluka), sodium azide (NaN3; Alfa Aesar), 4-(phenyldiazenyl) phenol (Acros Organic, USA), 6-chloro-1-hexanol (Acros Organic, 97%), DLdithiothreitol (DTT, Aldrich, 99%), p-toluenesulfonyl chloride (TsCl, 99%, Acros Organic) and 3,3′-dithiodipropionic acid (DTDP, Aldrich, 99%) were used as received. 2,2′-azobisisobutyronitrile (AIBN; Aldrich, 98%) was recrystallized from ethanol. 2-(2methoxyethoxy)ethyl methacrylate (MEO2MA) and oligo(ethylene glycol) methacrylate (OEGMA, Mn = 475 g/mol) was purchased from Aldrich and passed through a column of activated basic alumina to remove inhibitors. Methylene dichloride (CH2Cl2), tetrahydrofuran (THF), N,N-dimethylformamide (DMF) and triethylamine (Et3N) were dried over CaH2 and distilled under reduced pressure. Propargyl 2-bromoisobutyrate (PBiB) were synthesized according to the methods in the literature [43]. 2.2. Characterization 2.2.1. Nuclear magnetic resonance spectroscopy (NMR) 1 H NMR spectrum was obtained from a Bruker AV 400 NMR spectrometer at ambient temperature with CDCl3, D2O and DMSO-d6 as the solvent. The chemical shifts were relative to tetramethylsilane. 2.2.2. Attenuated total internal reflectance fourier transform infrared (ATR FT-IR) ATR FT-IR spectra of samples were recorded on an Equinoss/Hyperion2000 spectrometer (Bruker, Germany). 2.2.3. Transmission electron microscopy (TEM) The TEM images were obtained using an H-800 (Hitachi, Japan) TEM at an accelerating voltage of 120 kV. The samples for TEM observation were prepared by dropping 10 μL of polymer solutions on copper grids coated with thin films and carbon. 2.2.4. Fluorescence spectroscopy Fluorescence spectra were recorded using an F-2500 spectrometer (Hitachi, Japan) with a xenon lamp source. Fluorescence scans were performed at room temperature in the range of 340–600 nm using increment of 1 nm with the excitation wavelength of 335 nm. The slit widths were set at 5 nm for both the excitation and the emission. 2.2.5. Optical measurement The transmittance of the supramolecular assembly solutions (5.0 mg/mL) at various temperatures was measured at 500 nm on a UV–Vis spectrophotometer (U-3310, Hitachi, Japan) fitted with the temperature controller. Sample cell was thermostated at different temperatures ranging from 25 to 50 °C prior to measurements using the temperature controller. The lower critical solution temperature (LCST) was defined as the temperature producing a 50% decrease in optical transmittance. 2.2.6. UV and visible light irradiation A UV LED irradiator (UVATA, λ0 = 365 nm) and a Vis LED irradiator (CCS, λ0 = 450 nm) were used to induce the photoisomerization of Azo moieties. 2.2.7. Dynamic light scattering (DLS) DLS studies were conducted using a Zetasizer Nano ZS90 instrument (Malvern Instruments) equipped with a multipurpose 398
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autotitrator (MPT-2) at a fixed scattering angle of 90°. The data were processed by cumulants analysis of the experimental correlation function. The hydrodynamic diameters (Dh) were calculated from the computed diffusion coefficient using the Stokes-Einstein equation. 2.3. Synthesis of the host polymer β-CD-P(MEO2MA-co-OEGMA) The synthetic strategy of the host polymer β-CD-P(MEO2MA-co-OEGMA) is illustrated in Scheme 1a. 2.3.1. Synthesis of β-CD-OTs β-CD (50 g, 0.04 mol) was dissolved into 500 mL of 0.4 M sodium hydroxide (8 g, 0.2 mol) solution under stirring, and then 35 g of p-toluenesulfonyl chloride was added in at a very low rate to ensure that the substitution occurred at the C6 position. The mixture was stirred at 5 °C for 30 min. The obtained suspension was filtered and concentrated hydrochloric acid (20 g) was added into the filtrate solution to neutralize the pH value of the solution. The resultant solution was stirred for 1 h. The precipitation was collected by vacuum filtration and washed by water three times. Finally, the product was dried to constant weight in a vacuum oven at room temperature for 48 h (yield: 78%). 1H NMR (DMSO-d6, δ, ppm): 2.45 (s, 3H, -CH3), 3. 3.23–3.38 (m, 14H, H-2,4), 3.60–3.75 (m, 28H, H-3,5,6), 4.79 (d, 2H, H-1), 4,86 (t, 5H, H-1), 5.74 (m, 14H, OH-2,3), 7.45 (d, 2H, H-9), 7.77 (d, 2H, H-8). 2.3.2. Synthesis of Mono-6-deoxy-6-azido-β-cyclodextrin (β-CD-N3) 15.0 g (0.0116 mol) of β-CD-OTs was dissolved in 150 mL water and heated to 80 °C. 4.7 g (0.0723 mol) of sodium azide was added to this solution. After stirring 6 h at 105 °C, the clear solution was precipitated in 900 mL acetone. The white precipitate was isolated by filtering and dried over MgSO4 (yield: 82%). 1H NMR (DMSO-d6, δ, ppm): 3.23–3.38 (m, 14H, H-2,4), 3.65 (m, 28H, H3,5,6), 4.79 (d, 2H, H-1), 4,86 (t, 5H, H-1), 5.74 (m, 14H, OH-2,3). 2.3.3. Synthesis of bromide modified β-CD (β-CD-Br) β-CD-N3 (6.02 g, 5.19 mmol), PBiB (2.07 g, 12.94 mmol), and PMDETA (305 μL, 1.5 mmol) were dissolved in 50 mL dry DMF. After one freeze-pumpthaw cycle, CuBr (0.738 g, 5.27 mmol) was introduced under the protection of argon flow. The reaction flask was degassed by three freeze-pumpthaw cycles and then placed in an oil bath stirring at 40 °C for 48 h. The reaction mixture was then exposed to air, and precipitated in 600 mL ethanol. The precipitation was obtained by suction filtration and washed three times with ethanol, yielding a light blue solid dried overnight in a vacuum oven (yield: 78%). 1H NMR (DMSO-d6, δ, ppm): 1.86 (s, 6H, -COOC(CH3)2Br), 3.34 (br, 14H, H-2,4), 3.65 (br, 28H, H-3,5,6), 4.52 (br, 6H, OH-6), 4.84 (d, 7H, H-1), 5.03 (s, 2H, -CH2-O-), 5.73 (br, 14H, OH-2,3), 8.03–8.15 (d, 2H, -CH- of the 1, 2, 3-triazole group). 2.3.4. Synthesis of the host polymer β-CD-P(MEO2MA-co-OEGMA) β-CD-P(MEO2MA-co-OEGMA) was synthesized by ATRP of MEO2MA monomer and OEGMA monomer with β-CD-Br as the initiator and CuBr/PMDETA as the catalyst system. A dried Schlenk flask with a magnetic stirrer was charged with β-CD-Br (0.6 g, 0.44 mmol), MEO2MA (7.38 g, 39.25 mmol), OEGMA (1.62 g, 3.407 mmol), CuBr (63.07 mg, 0.44 mmol) and anhydrous DMF (6 mL). The flask was degassed with three freeze-evacuate-thaw cycles. PMDETA (92.86 μL, 0.44 mmol) was deoxygenated by bubbling dry argon before injection into the reaction system by syringe. The polymerization reaction was performed at 80 °C for 4 h. After being cooled to room temperature, the reaction flask was opened to air, and the crude product was diluted with THF and passed through a neutral oxide alumina column to remove the copper catalysts. Then the filtered solution was purified by dialysis (molecular weight cut-off: 3000 Da) against water to remove the catalyst and unreacted monomers. After lyophilization to remove the water, the final purified product was obtained (yield: 83%). Mn, GPC = 12,680 g/mol, Mw/Mn = 1.26. Mn, NMR = 14,000 g/mol. 1H NMR (DMSO-d6, δ, ppm): 0.58–1.24 (m, 3H, CH2C(CH3)), 1.59–2.21 (m, 2H, CH2C(CH3)), 3.39 (s, 3H, OCH2CH2OCH3), 3.48–3.79 (t, 4H, OCH2CH2O), 4.09 (t, 2H, COOCH2CH2O), 5.03 (s, 2H, -CH2-O-), 5.73 (m, 14H, OH-2,3), 8.13 (s, 1H, -CH- of the 1, 2, 3-triazole group). 2.4. Synthesis of the guest polymer Azo-PCL-SS-PEG The synthetic strategy of the guest polymer Azo-PCL-SS-PEG is illustrated in Scheme 1b. 2.4.1. Synthesis of Azo-OH The 6-(4-(phenyldiazenyl) phenoxy) hexan-1-ol (Azo-OH) was prepared using the similar procedures according to the literature [44]. 4-(phenyldiazenyl) phenol (4.95 g, 25 mmol), 6-chloro-1-hexanol (5.1 g, 37.5 mmol), potassium carbonate (5.175 g, 37.5 mmol) and a trace amount of potassium iodide were dissolved in 100 mL DMF. The mixture solution was refluxed for 12 h and washed with a large amount of water. The product was extracted with chloroform, dried by anhydrous MgSO4. After the solvent was removed by evaporation, the crude product was purified by recrystallization from ethanol to obtain the purified product (yield: 35%). 1H NMR (CDCl3, δ, ppm): 1.44–1.54 (m, 4H, -CH2-CH2-CH2-CH2-OH), 1.63(m, 2H, -CH2-CH2-OH), 1.84(m, 2H, -Ph-O-CH2CH2-), 3.67 (t, 2H, -CH2-OH), 4.05 (t, 2H, -Ph-O-CH2-), 7.00–7.86 (m, 9H, -CH- of the azobenzene group). 2.4.2. Synthesis of Azo-PCL-OH Azo-PCL-OH was synthesized by ROP of CL with Azo-OH as the initiator. Briefly, CL (13.389 g, 0.1175 mol), Azo-OH (1 g, 399
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3.34 mmol), and a catalytic amount of Sn(Oct)2 (140 mmol) were added to a fire-dried polymerization tube quickly. The tube was then connected to a Schlenk line, where exhausting-refilling processes were repeated thrice. The tube was put into an oil bath at 120 °C under argon atmosphere with stirring and cooled to room temperature after polymerization for 24 h. The resulting product was dissolved in chloroform and precipitated twice in methanol. The purified polymer was dried in vacuum at room temperature (yield: 95%). Mn, NMR = 4860 g/mol. 1H NMR (CDCl3, δ, ppm): 1.40 (m, 2H, CH2CH2CH2CH2CH2O), 1.65 (m, 4H, CH2CH2CH2CH2CH2O), 2.30 (t, 2H, CH2CH2CH2CH2CH2O), 4.05 (t, 2H, CH2CH2CH2CH2CH2O), 7.00–7.86 (m, 9H, -CH- of the azobenzene group). 2.4.3. Synthesis of Azo-PCL-SS-COOH Azo-PCL-OH (5 g, 1.166 mmol), DTDP (0.245 g, 11.7 mmol), DCC (0.72 g, 3.5 mmol), DMAP (0.2134 g, 1.75 mmol) and THF (25 mL) were added into a 50 mL around-bottomed flask. The reaction was carried out at room temperature for 36 h under argon atmosphere with stirring. The reaction byproduct dicyclohexylcarbodiurea (DCU) was removed by vacuum filtration. The solution was concentrated by rotary evaporator and precipitated in methanol. The product was dried to constant weight in a vacuum oven at room temperature for 48 h (yield: 91%). Mn, NMR = 5050 g/mol. 1H NMR (CDCl3, δ, ppm):1.40 (m, 2H, CH2CH2CH2CH2CH2O), 1.65 (m, 4H, CH2CH2CH2CH2CH2O), 2.30 (t, 2H, CH2CH2CH2CH2CH2O), 2.9 (t, 2H, SSCH2CH2COOH), 3.0 (t, 2H, SSCH2CH2OC(O)), 4.05 (t, 2H, CH2CH2CH2CH2CH2O), 7.00–7.86 (m, 9H, -CH- of the azobenzene group). 2.4.4. Synthesis of Azo-PCL-SS-PEG Azo-PCL-SS-COOH (2 g, 4.44 mmol), MPEG (2.2 g, 4.44 mmol), DMAP (0.195 g, 6.66 mmol) and DCC (0.275 g, 13.32 mmol) were dissolved in 25 mL CH2Cl2. And the reaction was carried out at room temperature for 48 h under vigorous stirring. The reaction byproduct dicyclohexylcarbodiurea (DCU) was removed by vacuum filtration. After the CH2Cl2 solvent was removed by evaporation, the crude product was then dialyzed against distilled water for 72 h using a dialysis bag (molecular weight cut-off: 8000–14,000 Da) to remove unreacted stuffs. After lyophilization to remove the water, the final purified product was obtained (yield: 85%). Mn, 1 GPC = 10,320 g/mol, Mw/Mn = 1.24. Mn, NMR = 11,100 g/mol. H NMR (CDCl3, δ, ppm):1.40 (m, 2H, CH2CH2CH2CH2CH2O), 1.65 (m, 4H, CH2CH2CH2CH2CH2O), 2.30 (t, 2H, CH2CH2CH2CH2CH2O), 3.65 (t, 4H, OCH2CH2O), 4.05 (t, 2H, CH2CH2CH2CH2CH2O), 7.00–7.86 (m, 9H, -CH- of the azobenzene group). 2.5. Preparation of the supramolecular assemblies The Azo-PCL-SS-PEG (22.2 mg) and β-CD-P(MEO2MA-co-OEGMA) (28 mg) were dissolved in DMF (10 mL). It was then mixed manually followed by sonication for 30 min. Then the mixed solution was dialyzed against distilled water for 72 h using a dialysis bag (molecular weight cut-off: 8000–14,000 Da) to obtain the supramolecular assemblies. 2.6. Critical micelle concentration (CMC) measurement of the supramolecular assemblies in aqueous solutions The critical micelle concentration (CMC) was measured using pyrene as a fluorescence probe. 5 mg pyrene was dissolved in 10 mL acetone and then 10 μL of the solution was added into each cuvette. The acetone was allowed to evaporate for 2 h at room temperature. Then 2.0 mL of aqueous supramolecular assembly solution ranging from 1.95 mg/L to 1000 mg/L were added into the pyrene-containing cuvette separately. Upon sonication for 10 min, the solutions were kept at room temperature and equilibrated for 24 h before fluorescent emission measurements with the excitation wavelength of 335 nm. The spectra were recorded in the 340–600 nm wavelength range. For each spectrum obtained, the intensity ratio of the first and third peaks, I1/I3, was calculated. The CMC was estimated as the concentration at which I1/I3 began to drop, suggesting that the micellation of the polymer occurred. 3. Results and discussion 3.1. Synthesis of the host polymer β-CD-P(MEO2MA-co-OEGMA) The products of β-CD-OTs and β-CD-N3 were characterized by 1H NMR (Fig. 1a and b) and FT-IR (Fig. 2b and c). As shown in Fig. 1a and b, all the protons signals assigned to β-CD-OTs and β-CD-N3 could be clearly observed, indicating the successful preparation of the pure β-CD-OTs and β-CD-N3. Moreover, a comparison of the FT-IR spectra of β-CD-OTs and β-CD-N3 in Fig. 2b and c revealed the appearance of a strong absorbance peak at 2100 cm−1 for β-CD-N3, which was characteristic absorption peak of the azide group, further confirming the successful synthesis of β-CD-N3. β-CD-Br was obtained via the click reaction of β-CDN3 and excess PBiB. As shown in the Fig. 1c, the peaks at about 8.1 of methine proton in 1,2,3-triazole ring (peak a) proved the formation of the functional initiator β-CD-Br. As shown in the Fig. 2d, after the click reaction, the absorption band at 2100 cm−1 disappeared, and the peck of carbonyl group at 1730 cm−1 occurred, indicating the occurrence of click reaction of β-CD-N3 and PBiB. β-CD-P(MEO2MA-co-OEGMA) was synthesized by ATRP of MEO2MA and OEGMA with β-CD-Br as initiator and CuBr/PMDETA as catalyst system. The feed ratio of MEO2MA and OEGMA was 92:8. The peaks of protons from MEO2MA and OEGMA were corresponded to the peaks of f and g in Fig. 1d. All characteristic signals of the host polymer β-CD-P(MEO2MA-co-OEGMA) could be observed and the corresponding peaks have been assigned. And the number-average molecular weight calculated by 1H NMR (Mn, NMR) was 14,000 g/mol. 400
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Fig. 1. 1H NMR spectrum of (a) β-CD-OTs, (b) β-CD-N3, (c) β-CD-Br, and (d) β-CD-P(MEO2MA-co-OEGMA).
Fig. 2. FT-IR spectra of (a) β-CD, (b) β-CD-OTs, (c) β-CD-N3 and (d) β-CD-Br.
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Fig. 3. 1H NMR spectrum of (a) Azo-OH, (b) Azo-PCL-SS-COOH, (c) Azo-PCL-OH, and (d) Azo-PCL-SS-PEG.
3.2. Synthesis of the guest polymer Azo-PCL-SS-PEG Azo-OH was synthesized by the Williamson etherification of 4-(phenyldiazenyl) phenol and 6-chloro-1-hexanol. Fig. 3a shows the H NMR spectrum of Azo-OH. All the protons of Azo-OH could be observed. The integral area ratio of peak e (4.08 ppm) to peak c (7.95 ppm) was 1: 2, indicating the successful synthesis of Azo-OH. Azo-PCL-OH was synthesized by ROP of CL with the obtained Azo-OH as the initiator. The 1H NMR spectrum of Azo-PCL-OH is shown in Fig. 3b. All the proton signals of the polymer could be clearly detected. The degree of polymerization of CL was obtained from the integration of the proton signals at 4.05 ppm (peak m) to that at 3.65 ppm (peak m’), which was about 35. Azo-PCL-SS-COOH was synthesized by DCC reaction. The 1H NMR spectrum of AzoPCL-SS-COOH is shown in Fig. 3c. The peaks of protons from Azo and PCL segments can be clearly observed. According to the proton peaks of p and q, it can be found that the disulfide bond of 3,3′-dithiodipropionic acid was successfully attached to the polymer. The Azo-PCL-SS-PEG copolymer was prepared through the esterification reaction of Azo-PCL-SS-COOH with MPEG. From the 1H NMR spectrum of Azo-PCL-SS-PEG (Fig. 3d), the proton peak i (3.6 ppm) and peak j (3.4 ppm) could be well assigned to PEG. And the number-average molecular weight calculated by 1H NMR (Mn, NMR) was 11,100 g/mol. The GPC traces of β-CD-P(MEO2MA-co-OEGMA) and Azo-PCL-SS-PEG are shown in Fig. 4. The traces were mono-modal, indicating that the pure polymers were obtained. The number-average molecular weights (Mn, GPC) of β-CD-P(MEO2MA-co-OEGMA) and Azo-PCL-SS-PEG were 12,680 g/mol and 10,320 g/mol, respectively. 1
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Fig. 4. GPC traces of β-CD-P(MEO2MA-co-OEGMA) and Azo-PCL-SS-PEG.
3.3. Self-assembly of the supramolecular polymer Resulting from the amphiphilic property, the supramolecular polymer could self-assemble into spherical micelles in aqueous solutions. Fig. 5a shows the TEM images of the supramolecular assemblies. It can be seen that the supramolecular assemblies had a regular spherical morphology with a diameter of ∼85 nm, indicating that they were micelles. Consistently, the hydrodynamic diameter (Dh) of the supramolecular assemblies determined using DLS (Fig. 5b) was 109.85 nm, and the particle dispersion index (PDI) was 0.313. The CMC of the supramolecular polymer in aqueous solutions was measured using pyrene as the fluorescent probe, and the CMC of the supramolecular polymer was 81.8 mg/L as shown in Fig. 5c. 3.4. UV light -responsive behaviors of the supramolecular assemblies Due to the UV-response of the β-CD/Azo inclusion complex, the supramolecular assemblies demonstrated UV-responsive properties in aqueous solution. Fig. 6a shows the TEM images of the supramolecular assemblies after UV light irradiation. The size of the supramolecular assemblies decreased from ∼85 nm to ∼45 nm after UV irradiation, and the Dh value changed from 109.85 nm
Fig. 5. (a) TEM image and (b) DLS data of the supramolecular assemblies at 25 °C (Concentration: 0.5 mg/mL), (c) CMC measurement of the supramolecular polymer.
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Fig. 6. (a) TEM image and (b) DLS data of the supramolecular assemblies after UV irradiation at 365 nm for 1 min (Concentration: 0.5 mg/mL).
to 51.75 nm with a PDI of 0.526 (Fig. 6b). This was because the trans-Azo changed to cis-state upon UV light irradiation, and then departed from the cavity of β-CD. As a result, the hydrophilic-hydrophobic balance of the system altered, leading to the decrease of the size of spherical micelles. The light-response of the supramolecular assemblies was further traced and confirmed by UV–vis spectra. The absorption bands at 355 and 440 nm are ascribed to the π-π∗ transition of the trans-Azo form and n-π∗ transition of the cis-Azo form of the Azo group, respectively. As shown in Fig. 7a, upon irradiation by UV light, the absorption peak at 355 nm was remarkably decreased, while the absorption at 440 nm was slightly increased, indicating the photo-isomerization of the trans-Azo to cis-Azo. When the same sample was irradiated with visible light, the peak at 355 nm increased remarkably, and the peak at around 440 nm decreased concomitantly, which indicated the reversible isomerization of Azo groups from cis to trans state (Fig. 7b). Furthermore, on alternating irradiation of the sample with UV and visible light, the reversible trans to cis photo-isomerization process could be recycled many times (Fig. 7c). Fig. 7d shows the TEM images of the supramolecular assemblies after four UV–visible light irradiation cycles. The supramolecular assemblies returned to the initial state with the size of ∼90 nm. The Dh value of the supramolecular assemblies was 112.26 nm with a PDI was 0.313 (Fig. 7e).
Fig. 7. UV–Vis spectrum of supramolecular assemblies: (a) 365 nm UV irradiation induced trans-to-cis transition, (b) 450 nm visible light irradiation induced cis-totrans transition, (c) changes of Azo-CD inclusion upon alternating irradiation with 365 nm UV light and 450 nm visible light, (d) TEM image and (e) DLS data of the supramolecular assemblies after four UV–visible light irradiation cycles at 25 °C (Concentration: 0.5 mg/mL).
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Fig. 8. (a) Optical transmittance and (b) temperature dependence of hydrodynamic diameter (Dh) of the supramolecular assemblies, (c) TEM image and (d) DLS data of the supramolecular assemblies after heating to 50 °C (Concentration: 0.5 mg/mL).
3.5. Thermo-responsive behaviors of the supramolecular assemblies Benefitting from the thermo-response of P(MEO2MA-co-OEGMA) segments, the supramolecular assemblies presented thermoresponsive properties. Fig. 8a shows the transmittance curve of the supramolecular assembly solution. It can be seen that the transmittance curve showed a sharp decreasing transition during heating from 38 °C to 40 °C and the lower critical solution temperature (LCST) value was calculated to be ∼38.8 °C. The Dh values of the supramolecular assemblies in aqueous solution as a function of temperature are shown in Fig. 8b. At relatively lower temperatures, the Dh values were small and changed little. In contrast, when at higher temperature ranges (> 37 °C), the Dh values increased significantly with the rise of temperature, which was due to the aggregation among micelles. The reason for the thermo-response of the supramolecular assemblies in aqueous solutions was that P(MEO2MA-co-OEGMA) chains existed in random coil conformations at low temperature range due to the hydrogen-bonding reaction between the ether oxygen and water molecules. When the temperature increased to a critical value (> LCST), P(MEO2MAco-OEGMA) chains shrank into a globular structure and became hydrophobic because the hydrogen bonds between the ether oxygen of P(MEO2MA-co-OEGMA) and water molecules collapsed. As a result, the intermolecular hydrophobic attractions were thermodynamically favoured and the supramolecular assemblies tended to agglomerate, leading to the increase in Dh and visible turbidity. Fig. 8c shows the TEM image of supramolecular assemblies after heating to the temperature of 50 °C. It can be observed that the spherical micelles became smaller and aggregated with each other. At the same time, the DLS trace of the heated solution was asymmetric with a shoulder and the PDI value was 0.813 as shown in Fig. 8d, indicating that the aggregates were irregular. The thermo-response of the supramolecular assemblies was further confirmed using 1H NMR spectra in D2O. As shown in Fig. 9, the chemical shifts in P(MEO2MA-co-OEGMA) could be observed and their intensities decreased with increasing temperature. This decrease was because that P(MEO2MA-co-OEGMA) segments underwent a configuration transition from a random coil conformation to a globular structure and the P(MEO2MA-co-OEGMA) solubility decreased after the collapse of the hydrogen-bonding interaction between the ether oxygen and water molecules upon heating. It can be seen that peak d was remained in the spectra. This was because at high temperatures the water-soluble PEG segments of the guest polymer were stretched out in the solution to stabilize the micelles despite the P(MEO2MA-co-OEGMA) segments were hydrophobic.
3.6. Redox-responsive behaviors of the supramolecular assemblies Due to the existence of the disulfide bonds, the supramolecular assemblies presented redox responses. Fig. 10a shows the TEM image of assemblies treated with 10 mM DTT over 24 h. It can be seen that the regular spherical micelles changed into irregular 405
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Fig. 9. 1H NMR spectra of the supramolecular polymer in D2O conducted at different temperatures.
aggregates with different sizes, and the Dh of assemblies increased to 215.06 nm with a wide PDI of 0.743 after adding DTT (Fig. 10b). The reason for this change was that the disulfide bonds linking the PCL and PEG blocks were broken in the reaction with DTT. As the reductive cleavage of the disulfide bonds occurred, the PEG segments would shed. Then the hydrophilic-hydrophobic balance of supramolecular assemblies was broken and the micelles became unstable, and then the irregular aggregates were formed.
4. Conclusions In summary, novel UV light-, thermo- and redox-responsive supramolecular micelles consisted of β-CD-P(MEO2MA-co-OEGMA)/ Azo-PCL-SS-PEG have been successfully prepared based on the inclusion complexation between β-CD and Azo. The conformation and morphology of the supramolecular micelles could be adjusted by UV irradiation, temperature and redox potential. Alternating irradiation of the solution with UV or visible light could induce the reversible supramolecular self-assembly and disassembly of micelles. When the temperatures were higher than a critical value, the micelles became smaller and aggregated with each other. After adding DTT into the micellar system, the spherical micelles changed into irregular and became smaller. Therefore, the size and morphology of the supramolecular assemblies could be fine-tuned to each stimulus independently. As a result, the supramolecular assemblies with controllable size and morphology may have potential applications in nanotechnology and smart-material fields.
Acknowledgment The authors gratefully acknowledge the financial support of the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry and the National Key Technology R & D Program (no. 2012BAI15B06).
Fig. 10. (a) TEM image, and (b) DLS data of the supramolecular assemblies after addition of DTT (10 mM) over 24 h (Concentration: 0.5 mg/mL).
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