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Communication
Synthesis, Self-Assembly, and Multi-Stimuli Responses of a Supramolecular Block Copolymer Weizhong Yuan,* Jinju Wang, Lulin Li, Hui Zou, Hua Yuan, Jie Ren
A supramolecular block copolymer is prepared by the molecular recognition of nucleobases between poly(2-(2-methoxyethoxy)ethyl methacrylate-co-oligo(ethylene glycol) methacrylate)SS-poly(ε-caprolactone)-adenine (P(MEO2MA-co-OEGMA)-SS-PCL-A) and uracil-terminated poly(ethylene glycol) (PEG-U). Because the block copolymer is linked by the combination of covalent (disulfide bond) and noncovalent (A U) bonds, it not only has similar properties to conventional covalently linked block copolymers but also possesses a dynamic and tunable nature. The copolymer can self-assemble into micelles with a PCL core and P(MEO2MA-co-OEGMA)/PEG shell. The size and morphologies of the micelles/aggregates can be adjusted by altering the temperature, pH, salt concentration, or adding dithiothreitol (DTT) to the solution. The controlled release of Nile red is achieved at different environmental conditions.
1. Introduction Considerable attention has been paid to stimuli-responsive polymers and their self-assembled structures such as micelles and vesicles due to their prospective applications in nanotechnology, drug-delivery, genetransport systems, recyclable catalysis, and separations.[1] These polymeric self-assembled structures can undergo reversible changes in response to external stimuli such as temperature, pH, redox, light, salt, sugar, and carbon dioxide.[2–4] During the past two decades,
Prof. W. Yuan, J. Wang, L. Li, H. Zou, Prof. H. Yuan, Prof. J. Ren Institute of Nano and Bio-polymeric Materials, School of Materials Science and Engineering, Tongji University, 4800 Cao’an Road, Shanghai 201804 , China E-mail: [email protected] Prof. W. Yuan, Prof. J. Ren Key Laboratory of Advanced Civil Materials, Ministry of Education, 4800 Cao’an Road, Shanghai 201804 , China
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numerous stimuli-responsive polymeric micelles have been reported.[5] Among thermoresponsive micelles, those based on amphiphilic copolymers containing 2-(2-methoxyethoxy)ethyl methacrylate (MEO2MA) and oligo(ethylene glycol) methacrylate (OEGMA) are very typical.[6,7] Random copolymers of MEO2MA and OEGMA, P(MEO2MA-co-OEGMA), belong to the poly(ethylene glycol)-based copolymers and exhibit a critical phase transition temperature in water that can be finely tuned between 26 and 90 °C by altering the ratio of MEO2MA and OEGMA.[8–10] You and co-workers[11] reported biocompatible nanocapsules with temperature-responsive properties based on P(MEO2MA-co-OEGMA) and found that the lower critical solution temperature (LCST) of the nanocapsules can be tuned precisely. The model drug fluorescein isothiocyanate, which was encapsulated into the nanocapsules, demonstrated a controlled release behavior through altering the temperature. The disulfide bond is a typical redox-responsive chemical group, which is broken in the presence of DL-dithiothreitol (DTT) and glutathione (GSH).[12,13] Zhong et al.[14] reported the
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DOI: 10.1002/marc.201400308
Synthesis, Self-Assembly, and Multi-Stimuli Responses of a Supramolecular Block Copolymer
Macromolecular Rapid Communications www.mrc-journal.de
synthesis of reversibly shielded DNA polyplexes based on PDMAEMA-SS-PEG-SS-PDMAEMA block copolymers. Adding DTT solution to the polyplexes triggered the breaking of disulfide bonds and the release of DNA. Compared with conventional covalently linked polymers, supramolecular polymers based on noncovalent interactions are more sensitive to external stimuli, which offers a novel strategy for the design of drug delivery systems with rapid response abilities.[15,16] Among these noncovalent interactions, complementary hydrogen bonds from nucleobases, which are moderately strong, highly directional, and sensitive to acidic pH and salt, show great potential in biomedical applications. Zhu and co-workers[17] reported supramolecular copolymer micelles based on complementary multiple hydrogen bonds of nucleobases for drug delivery and found that the release of doxorubicin was significantly faster at a mildly acid pH of 5.0 compared to the physiological pH. They also constructed supramolecular amphiphilic multi-arm hyperbranched and brush copolymers through the molecular recognition of nucleobases. Investigation showed that these micelles possessed many favorable traits, such as low cytotoxicity and excellent biodegradability, adequate drug loading capacity, and rapid drug release in response to the intracellular level of pH and salt concentration.[18] However, a majority of stimuli-responsive micelles reported up to now focus on single or dual stimuli, such as temperature/pH,[19] thiol/pH,[20] light/temperature,[21] and light/redox[22] responsive micelles. Multi-stimuli responsive micelles are rarely reported,[23,24] especially for multi-responsive micelles.[25] Multi-stimuli responsive micelles can achieve more functions and be modulated through more parameters. They may provide a unique opportunity to fine-tune their response to each stimulus independently, as well as precisely regulate the release profile during the combined effect of multiple stimuli. In this article, a novel block copolymer was synthesized by the combination of ring-opening polymerization (ROP), atom transfer radical polymerization (ATRP), coupling reaction, and noncovalent interaction (Scheme S1, Supporting Information). The three blocks in the block copolymer were linked by one covalent bond (disulfide bond) and one noncovalent interaction (hydrogen bond of nucleobases). Due to the presence of hydrophobic PCL, hydrophilic PEG, thermoresponsive P(MEO2MA-coOEGMA), disulfide bond, and nucleobases of adenine– uracil (A–U), the block copolymer can self-assemble into micelles in water and exhibits thermo, redox, salt, and pH multi-stimuli responses. The self-assembly behavior, morphologies of micelles, and the controlled release for model drug molecules (Nile red) at different stimulus condition were investigated.
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2. Experimental Section All experimental details are provided in the Supporting Information.
3. Results and Discussion 3.1. Hydrogen Bonding Interactions Between P(MEO2MA-co-OEGMA)-SS-PCL-A and PEG-U The formation of complementary hydrogen bonds between P(MEO2MA-co-OEGMA)-SS-PCL-A (1H and 13C NMR spectra are shown in Figures S1, S2, Supporting Information) and PEG-U (1H NMR spectrum is shown in Figure S3, Supporting Information, GPC is shown in Figure S4, Supporting Information) was analyzed by variable-temperature fourier transform infrared (FTIR) spectroscopy (Figure S5, Supporting Information) and variable-temperature 1H NMR spectroscopy for the blend of two polymers with equivalent molar quantities of A and U in 1,1,2,2-tetrachloroethane-d2. The temperature dependence of the NH proton chemical shift of the P(MEO2MA-co-OEGMA)-SSPCL-A and PEG-U complex at 3.0% in 1,1,2,2-tetrachloroethane-d2 is shown in Figure 1. The chemical shift of the NH resonance moved upfield systematically from 8.41 to 8.11 ppm when the temperature increased from 25 to 60 °C. The gradual decrease in the NH resonance with the increase of temperature is attributed to the dissociation of the complementary hydrogen bonds. As soon as the solution was cooled from 60 to 25 °C, the NH resonance returned to its original position at 8.41 ppm. This suggests that the complementary hydrogen bonds generated between P(MEO2MA-co-OEGMA)-SS-PCL-A and PEG-U and the supramolecular block polymer is thermoreversible in 1,1,2,2-tetrachloroethane-d2. The 1H NMR spectra of the NH chemical shift of the P(MEO2MA-co-OEGMA)-SS-PCLA and PEG-U complex (A–U = 1:2 and 2:1) as a function of the temperature in 1,1,2,2-tetrachloroethane-d2 were also measured (Figure S6, Supporting Information). 3.2. Self-Assembly of P(MEO2MA-co-OEGMA)-SS-PCL-A– U–PEG Micelles and Multi-Responses As an amphiphilic block copolymer, P(MEO2MA-coOEGMA)-SS-PCL-A–U–PEG can form self-assembled micelles in aqueous solution (Scheme 1). The formation of supramolecular copolymer micelles was confirmed by critical micelle concentration (CMC) measurement (Figure S7, Supporting Information). It can be seen that the CMC is 0.015 mg mL−1, indicating that the micelles can be formed at much lower concentration in aqueous solution. In order to investigate the thermo, redox, salt, and pH multi-responses of the supramolecular micelles, both
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Figure 1. 1H NMR spectra of the NH chemical shift of the P(MEO2MA-co-OEGMA)-SSPCL-A and PEG-U complex (an equimolar ratio of A and U) as a function of the temperature in 1,1,2,2-tetrachloroethane-d2.
dynamic light scattering (DLS) and transmission electron microscopy (TEM) measurements were performed. The apparent hydrodynamic radius (Rh) and the size distribution of the micelles can be determined by DLS. Figure S8 (Supporting Information) shows the apparent Rh and size distributions of P(MEO 2 MA- co -OEGMA)-SS-PCL-A–U– PEG micelles after exposed to various stimuli: 25 °C, 40 °C, DTT (10 × 10−3 M), pH 7.4 (0.12 M Na+), pH 7.4 (0.56 M Na+), and pH 5.0 (0.12 M Na+). At 25 °C (in water), the hydrophilic P(MEO2MA-coOEGMA) and PEG segments are mainly in the shell of the self-assembled structures, whereas the hydrophobic PCL segments are mainly in the core. These selfassembled structures aggregated into stable micelles (Scheme 1). The content of the hydrophilic part is much higher than that of the hydrophobic part in the block copolymer, and the micelles have a narrow size distribution with an apparent Rh of 55.8 nm and with a PDI of 0.096. When the temperature increased to 40 °C, P(MEO2MA-co-OEGMA) gradually shrank into a globular structure from the random coil conformation,
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and became more hydrophobic. Therefore, at this condition, the PEG segments are mainly in the corona of the self-assembled structures, whereas the hydrophobic P(MEO2MA-co-OEGMA) and PCL segments are mainly in the core. The micelles tended to aggregate into compounding micelles or aggregates of larger size (Rh = 103.5 nm, PDI = 0.108). When DTT was added to the micelles solution at 25 °C, the disulfide bonds linking the P(MEO2MAco-OEGMA) and PCL blocks were broken in the reaction with DTT. As a result, the hydrophilic P(MEO2MA-co-OEGMA) blocks were shedded from the micelles. The micelles aggregated into aggregates of large size (Scheme 1). The apparent Rh increased to 238.4 nm. Because of the hydrogen bonding interaction between hydrophobic PCL-A and hydrophilic PEG-U, the supramolecular block copolymer micelles were unstable at acidic pH and high salt concentration. In order to evaluate the
Scheme 1. Schematic presentation of the micelle of P(MEO2MA-co-OEGMA)-SS-PCL-A– U–PEG and the multi-stimuli response.
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Synthesis, Self-Assembly, and Multi-Stimuli Responses of a Supramolecular Block Copolymer
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responsive ability, the micelles were treated with a different pH buffer and salt concentration and the sizes of the micelles were recorded by DLS measurement. As shown in Figure S8 (Supporting Information), in a 0.12 M salt solution at pH 7.4, the apparent Rh of the micelles (62.8 nm) shows a slight increase compared with that in deionized water. When the salt concentration increased to 0.56 M at pH 7.4, the apparent Rh of the micelles increased to 85.4 nm, which indicated that the hydrogen bonding interaction obviously decreased at high salt concentrations. The hydrophilic PEG was detached from the micelles, the hydrophobicity of the micelles increased. Therefore, aggregation of the micelles occurred to some degree. Aggregates with larger size formed (Scheme 1). When the pH was lowered to 5.0 (0.12 M), the apparent Rh of the micelles also increased to 104.1 nm. This can be explained by the increased protonation of the nitrogen atoms of the nucleobases and the subsequent detachment of hydrophilic PEG blocks, which caused the aggregation of micelles to form large aggregates. In order to further understand the thermoresponse of micelles, the change of the apparent Rh at different temperatures was investigated. Figure S9 (Supporting Information) shows the plot of the apparent Rh of the micelles in aqueous solution as a function of temperature. In the lower temperature ranges, the apparent Rh values are relatively small and change slightly. In contrast, the values increase significantly in the higher temperature ranges. For example, the apparent Rh of the micelles was about 59.2 nm at 31 °C, but the apparent Rh increased to about 146.2 nm at 42 °C. At low temperatures, the P(MEO2MAco-OEGMA) chains existed in random coil conformation owing to the hydrogen-bonding interaction between the polymer and the water molecules. When the temperature increased to a critical value, the polymer chains shrank into a globular structure because the hydrogen bonds between the ether oxygen of P(MEO2MA-co-OEGMA) chains and water molecules collapsed and the block became hydrophobic. Therefore, the hydrophilic–hydrophobic balance underwent obvious modifications and aggregation of the micelles occurred. The variable-temperature 1H NMR of P(MEO2MA-co-OEGMA)-SS-PCL-A– U–PEG in D2O is shown in Figure S10 (Supporting Information) and the result further confirmed the thermoresponse of the micelles. The morphologies of the micelles at different conditions are shown in Figure 2. It can be seen from Figure 2a that dispersed spherical micelles can be obtained at 25 °C in aqueous solutions. However, when the temperature increased to 45 °C, larger micelles and aggregates were observed. Similar phenomena can be found in Figure 2c. At acidic pH (5.0) and high salt concentration (0.56 M) conditions, the stability of the micelles decreased and the obvious aggregation occurred. After DTT was added
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Figure 2. TEM images of P(MEO2MA-co-OEGMA)-SS-PCL-A–U– PEG micelles after exposure to various stimuli: a) 25 °C; b) 45 °C; c) pH 5.0 (0.56 M Na+); d) DTT (10 mM) (Concentration: 1 mg mL−1).
into the solution system and stirred for 48 h in Ar atmosphere at 25 °C, the regular micelles could not be observed in Figure 2d, because the disulfide bonds linking the P(MEO2MA-co-OEGMA)block and PCL block was broken after the reaction with DTT. The serious modification of the hydrophilic–hydrophobic balance changed the morphology of the micelles, leading to the aggregation and precipitation of irregular micelles. Therefore, the TEM results further confirmed the thermo, redox, salt, and pHmulti-responsive properties of P(MEO2MA-co-OEGMA)-SSPCL-A–U–PEG micelles. 3.3. Multi-Controlled Release Properties of Supramolecular Micelles The controlled release properties of Nile red were investigated for different temperature, pH, salt concentration, and DTT solutions. As shown in Figure 3, there was a slow release of about 6.2% of the incorporated Nile red within 120 h in deionized water. In a 0.56 M salt at pH 7.4, about 30.4% Nile red was released in 120 h. A high salt concentration led to the dissociation of the hydrogen bonds and the modification of the hydrophilic–hydrophobic balance of the micelles, followed by a comparatively fast release of the loaded Nile red. When the micelles were dispersed in an acidic environment with a pH value of 5.0, however, Nile red molecules entrapped in the supramolecular block copolymer micelles would be quickly released with the breakage of PEG segments from the micelles and the modification of the hydrophilic–hydrophobic balance. In 0.12 M salt solution at pH 5.0, about 41.8% Nile red was released in 120 h. It can be seen that an acidic medium significantly accelerated the release of Nile red from the micelles. The fast release of Nile red from micelles in an
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acidic pH values, and high salt concentrations compared to low temperature, physiological pH, and low salt concentrations. In addition, adding DTT to the micelles solution would rapidly increase the release of Nile red. Compared with other block copolymer systems, this copolymer can conveniently achieve multi-stimuli responses and corresponding variations of the structure of the micelles. It can be envisaged that such a supramolecular block copolymer with a unique structure and thermo, redox, salt, and pH multi-stimuli responses has potential applications in novel smart drug-delivery systems and intelligent nanomaterial fields.
Supporting Information Figure 3. Release profiles of Nile red from P(MEO2MA-co-OEGMA)SS-PCL-A–U–PEG micelles by altering the temperature, pH value, Na+ concentration, and adding DTT.
acidic environment was likely due to the protonation of the amino group of the nucleobases and the shedding of the PEG blocks in the micelles shell in acidic conditions and the serious modification of the hydrophilic–hydrophobic balance of the micelles. Moreover, when the temperature of the micelles solution was heated to 45 °C the release of Nile red was about 37.0% in 120 h, which should be attributed to the hydrophilic P(MEO2MA-co-OEGMA) chains which became hydrophobic at this temperature leading to aggregation and deformation of the micelles. After adding DTT to the micelles solution, the release of Nile red reached about 69.0%. Under the reaction of DTT, lots of hydrophilic P(MEO2MA-co-OEGMA) chains were broken from the micelle shell and the serious modification of the hydrophilic–hydrophobic balance led to the serious deformation, aggregation, and destruction of the micelles, which was beneficial for the release of encapsulated Nile red molecules. As a result, multi-controlled release properties can be achieved by altering the temperature, pH, salt concentration, and adding DTT to the micelle solution.
4. Conclusions A novel type of supramolecular block copolymer, P(MEO2MA-co-OEGMA)-SS-PCL-A–U–PEG, with a thermoresponsive disulfide bond and molecular recognition of nucleobases was synthesized. The supramolecular block copolymer could self-assemble into temperature, redox, pH, and salt multi-responsive micelles with a low CMC in aqueous solution. Micelles of different size and morphologies were obtained by adjusting the hydrophilic–hydrophobic balance via altering the temperature, pH value, salt concentration, and adding DTT. These micelles were found to release Nile red more rapidly at high temperature,
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Supporting Information is available from the Wiley Online Library or from the author. Acknowledgements: The authors thank the financial support of the National Key Technology R&D Program (No. 2012BAI15B061), the National Basic Research Program of China (973 Program: 2011CB013805), and the National High Technology Research and Development Program of China (No. 2013AA032202). Received: May 30, 2014; Revised: July 5, 2014; Published online: September 5, 2014; DOI: 10.1002/marc.201400308 Keywords: micelles; self-assembly; stimuli-sensitive polymers; supramolecular structures
[1] X. J. Cai, C. Y. Dong, H. Q. Dong, G. M. Wang, G. M. Pauletti, X. J. Pan, H. Y. Wen, I. Mehl, Y. Y. Li, D. L. Shi, Biomacromolecules 2012, 13, 1024. [2] W. Z. Yuan, H. Zou, W. Guo, A. Wang, J. Ren, J. Mater. Chem. 2012, 22, 24783. [3] P. Schattling, F. D. Jochuma, P. Theato, Polym. Chem. 2014, 5, 25. [4] W. Z. Yuan, W. Guo, Polym. Chem. 2014, 5, 4259. [5] W. Tian, X. Y. Wei, Y. Y. Liu, X. D. Fan, Polym. Chem. 2013, 4, 2850. [6] Z. S. Ge, Y. M. Zhou, J. Xu, H. W. Liu, D. Y. Chen, S. Y. Liu, J. Am. Chem. Soc. 2009, 131, 1628. [7] P. J. Roth, T. P. Davis, A. B. Lowe, Macromolecules 2012, 45, 322. [8] J. F. Lutz, Ö. Akdemir, A. Hoth, J. Am. Chem. Soc. 2006, 128, 13046. [9] J. Park, M. Moon, M. Seo, H. Choi, S. Y. Kim, Macromolecules 2010, 43, 8304. [10] Y. Y. Jiang, X. L. Hu, J. M. Hu, H. Liu, H. Zhong, S. Y. Liu, Macromolecules 2011, 44, 8780. [11] Z. K. Wang, D. Wang, H. Wang, J. J. Yan, Y. Z. You, Z. G. Wang, J. Mater. Chem. 2011, 21, 15950. [12] S. Matsumoto, R. J. Christie, N. Nishiyama, K. Miyata, A. Ishii, M. Oba, H. Koyama, Y. Yamasaki, K. Kataoka, Biomacromolecules 2009, 10, 119. [13] J. Y. Liu, Y. Pang, W. Huang, X. H. Huang, L. L. Meng, X. Y. Zhu, Y. F. Zhou, D. Y. Yan, Biomacromolecules 2011, 12, 1567.
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[14] C. H. Zhu, M. Zheng, F. H. Meng, F. M. Mickler, N. Ruthardt, X. L Zhu, Z. Y. Zhong, Biomacromolecules 2012, 13, 769. [15] X. F. Ji, J. Y. Li, J. Z. Chen, X. D. Chi, K. L. Zhu, X. Z. Yan, M. M. Zhang, F. H. Huang, Macromolecules 2012, 45, 6457. [16] C. Wang, Z. Q. Wang, X. Zhang, Acc. Chem. Res. 2012, 45, 608. [17] D. L. Wang, X. Y. Huan, L. J. Zhu, J. Y. Liu, F. Qiu, D. Y. Yan, X. Y. Zhu, RSC Adv. 2012, 2, 11953. [18] D. L. Wang, H. Y. Chen, Y. Su, F. Qiu, L. J. Zhu, X. Y. Huan, B. S. Zhu, D. Y. Yan, F. L. Guo, X. Y. Zhu, Polym. Chem. 2013, 4, 85. [19] W. Cui, X. M. Lu, K. Cui, L. Y. Liu, Y. Wei, Q. H. Lu, Langmuir 2012, 28, 9413.
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[20] X. L. Xu, H. Li, S. Z. Luo, T. Liu, Y. Y. Jiang, S. Y. Liu, Polym. Chem. 2013, 4, 695. [21] E. Blasco, B. V. K. J. Schmidt, C. Bamer-Kowollik, M. Piñol, L. Oriol, Polym. Chem. 2013, 4, 4506. [22] D. H. Han, X. Tong, Y. Zhao, Langmuir 2012, 28, 2327. [23] A. Klaikherd, C. Nagamani, S. Thayumanavan, J. Am. Chem. Soc. 2009, 131, 4830. [24] H. Wu, J. Dong, C. C. Li, Y. B. Liu, N. Feng, L. P. Xu, X. W. Zhan, H. Yang, G. J. Wang, Chem. Commun. 2013, 49, 3516. [25] S. Guragain, B. P. Bastakoti, M. Ito, S. Yusa, K. Nakashima, Soft Matter 2012, 8, 9628.
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Communication
Synthesis, Self-Assembly, and Multi-Stimuli Responses of a Supramolecular Block Copolymer Weizhong Yuan,* Jinju Wang, Lulin Li, Hui Zou, Hua Yuan, Jie Ren
A supramolecular block copolymer is prepared by the molecular recognition of nucleobases between poly(2-(2-methoxyethoxy)ethyl methacrylate-co-oligo(ethylene glycol) methacrylate)SS-poly(ε-caprolactone)-adenine (P(MEO2MA-co-OEGMA)-SS-PCL-A) and uracil-terminated poly(ethylene glycol) (PEG-U). Because the block copolymer is linked by the combination of covalent (disulfide bond) and noncovalent (A U) bonds, it not only has similar properties to conventional covalently linked block copolymers but also possesses a dynamic and tunable nature. The copolymer can self-assemble into micelles with a PCL core and P(MEO2MA-co-OEGMA)/PEG shell. The size and morphologies of the micelles/aggregates can be adjusted by altering the temperature, pH, salt concentration, or adding dithiothreitol (DTT) to the solution. The controlled release of Nile red is achieved at different environmental conditions.
1. Introduction Considerable attention has been paid to stimuli-responsive polymers and their self-assembled structures such as micelles and vesicles due to their prospective applications in nanotechnology, drug-delivery, genetransport systems, recyclable catalysis, and separations.[1] These polymeric self-assembled structures can undergo reversible changes in response to external stimuli such as temperature, pH, redox, light, salt, sugar, and carbon dioxide.[2–4] During the past two decades,
Prof. W. Yuan, J. Wang, L. Li, H. Zou, Prof. H. Yuan, Prof. J. Ren Institute of Nano and Bio-polymeric Materials, School of Materials Science and Engineering, Tongji University, 4800 Cao’an Road, Shanghai 201804 , China E-mail: [email protected] Prof. W. Yuan, Prof. J. Ren Key Laboratory of Advanced Civil Materials, Ministry of Education, 4800 Cao’an Road, Shanghai 201804 , China
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numerous stimuli-responsive polymeric micelles have been reported.[5] Among thermoresponsive micelles, those based on amphiphilic copolymers containing 2-(2-methoxyethoxy)ethyl methacrylate (MEO2MA) and oligo(ethylene glycol) methacrylate (OEGMA) are very typical.[6,7] Random copolymers of MEO2MA and OEGMA, P(MEO2MA-co-OEGMA), belong to the poly(ethylene glycol)-based copolymers and exhibit a critical phase transition temperature in water that can be finely tuned between 26 and 90 °C by altering the ratio of MEO2MA and OEGMA.[8–10] You and co-workers[11] reported biocompatible nanocapsules with temperature-responsive properties based on P(MEO2MA-co-OEGMA) and found that the lower critical solution temperature (LCST) of the nanocapsules can be tuned precisely. The model drug fluorescein isothiocyanate, which was encapsulated into the nanocapsules, demonstrated a controlled release behavior through altering the temperature. The disulfide bond is a typical redox-responsive chemical group, which is broken in the presence of DL-dithiothreitol (DTT) and glutathione (GSH).[12,13] Zhong et al.[14] reported the
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DOI: 10.1002/marc.201400308
Synthesis, Self-Assembly, and Multi-Stimuli Responses of a Supramolecular Block Copolymer
Macromolecular Rapid Communications www.mrc-journal.de
synthesis of reversibly shielded DNA polyplexes based on PDMAEMA-SS-PEG-SS-PDMAEMA block copolymers. Adding DTT solution to the polyplexes triggered the breaking of disulfide bonds and the release of DNA. Compared with conventional covalently linked polymers, supramolecular polymers based on noncovalent interactions are more sensitive to external stimuli, which offers a novel strategy for the design of drug delivery systems with rapid response abilities.[15,16] Among these noncovalent interactions, complementary hydrogen bonds from nucleobases, which are moderately strong, highly directional, and sensitive to acidic pH and salt, show great potential in biomedical applications. Zhu and co-workers[17] reported supramolecular copolymer micelles based on complementary multiple hydrogen bonds of nucleobases for drug delivery and found that the release of doxorubicin was significantly faster at a mildly acid pH of 5.0 compared to the physiological pH. They also constructed supramolecular amphiphilic multi-arm hyperbranched and brush copolymers through the molecular recognition of nucleobases. Investigation showed that these micelles possessed many favorable traits, such as low cytotoxicity and excellent biodegradability, adequate drug loading capacity, and rapid drug release in response to the intracellular level of pH and salt concentration.[18] However, a majority of stimuli-responsive micelles reported up to now focus on single or dual stimuli, such as temperature/pH,[19] thiol/pH,[20] light/temperature,[21] and light/redox[22] responsive micelles. Multi-stimuli responsive micelles are rarely reported,[23,24] especially for multi-responsive micelles.[25] Multi-stimuli responsive micelles can achieve more functions and be modulated through more parameters. They may provide a unique opportunity to fine-tune their response to each stimulus independently, as well as precisely regulate the release profile during the combined effect of multiple stimuli. In this article, a novel block copolymer was synthesized by the combination of ring-opening polymerization (ROP), atom transfer radical polymerization (ATRP), coupling reaction, and noncovalent interaction (Scheme S1, Supporting Information). The three blocks in the block copolymer were linked by one covalent bond (disulfide bond) and one noncovalent interaction (hydrogen bond of nucleobases). Due to the presence of hydrophobic PCL, hydrophilic PEG, thermoresponsive P(MEO2MA-coOEGMA), disulfide bond, and nucleobases of adenine– uracil (A–U), the block copolymer can self-assemble into micelles in water and exhibits thermo, redox, salt, and pH multi-stimuli responses. The self-assembly behavior, morphologies of micelles, and the controlled release for model drug molecules (Nile red) at different stimulus condition were investigated.
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2. Experimental Section All experimental details are provided in the Supporting Information.
3. Results and Discussion 3.1. Hydrogen Bonding Interactions Between P(MEO2MA-co-OEGMA)-SS-PCL-A and PEG-U The formation of complementary hydrogen bonds between P(MEO2MA-co-OEGMA)-SS-PCL-A (1H and 13C NMR spectra are shown in Figures S1, S2, Supporting Information) and PEG-U (1H NMR spectrum is shown in Figure S3, Supporting Information, GPC is shown in Figure S4, Supporting Information) was analyzed by variable-temperature fourier transform infrared (FTIR) spectroscopy (Figure S5, Supporting Information) and variable-temperature 1H NMR spectroscopy for the blend of two polymers with equivalent molar quantities of A and U in 1,1,2,2-tetrachloroethane-d2. The temperature dependence of the NH proton chemical shift of the P(MEO2MA-co-OEGMA)-SSPCL-A and PEG-U complex at 3.0% in 1,1,2,2-tetrachloroethane-d2 is shown in Figure 1. The chemical shift of the NH resonance moved upfield systematically from 8.41 to 8.11 ppm when the temperature increased from 25 to 60 °C. The gradual decrease in the NH resonance with the increase of temperature is attributed to the dissociation of the complementary hydrogen bonds. As soon as the solution was cooled from 60 to 25 °C, the NH resonance returned to its original position at 8.41 ppm. This suggests that the complementary hydrogen bonds generated between P(MEO2MA-co-OEGMA)-SS-PCL-A and PEG-U and the supramolecular block polymer is thermoreversible in 1,1,2,2-tetrachloroethane-d2. The 1H NMR spectra of the NH chemical shift of the P(MEO2MA-co-OEGMA)-SS-PCLA and PEG-U complex (A–U = 1:2 and 2:1) as a function of the temperature in 1,1,2,2-tetrachloroethane-d2 were also measured (Figure S6, Supporting Information). 3.2. Self-Assembly of P(MEO2MA-co-OEGMA)-SS-PCL-A– U–PEG Micelles and Multi-Responses As an amphiphilic block copolymer, P(MEO2MA-coOEGMA)-SS-PCL-A–U–PEG can form self-assembled micelles in aqueous solution (Scheme 1). The formation of supramolecular copolymer micelles was confirmed by critical micelle concentration (CMC) measurement (Figure S7, Supporting Information). It can be seen that the CMC is 0.015 mg mL−1, indicating that the micelles can be formed at much lower concentration in aqueous solution. In order to investigate the thermo, redox, salt, and pH multi-responses of the supramolecular micelles, both
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Figure 1. 1H NMR spectra of the NH chemical shift of the P(MEO2MA-co-OEGMA)-SSPCL-A and PEG-U complex (an equimolar ratio of A and U) as a function of the temperature in 1,1,2,2-tetrachloroethane-d2.
dynamic light scattering (DLS) and transmission electron microscopy (TEM) measurements were performed. The apparent hydrodynamic radius (Rh) and the size distribution of the micelles can be determined by DLS. Figure S8 (Supporting Information) shows the apparent Rh and size distributions of P(MEO 2 MA- co -OEGMA)-SS-PCL-A–U– PEG micelles after exposed to various stimuli: 25 °C, 40 °C, DTT (10 × 10−3 M), pH 7.4 (0.12 M Na+), pH 7.4 (0.56 M Na+), and pH 5.0 (0.12 M Na+). At 25 °C (in water), the hydrophilic P(MEO2MA-coOEGMA) and PEG segments are mainly in the shell of the self-assembled structures, whereas the hydrophobic PCL segments are mainly in the core. These selfassembled structures aggregated into stable micelles (Scheme 1). The content of the hydrophilic part is much higher than that of the hydrophobic part in the block copolymer, and the micelles have a narrow size distribution with an apparent Rh of 55.8 nm and with a PDI of 0.096. When the temperature increased to 40 °C, P(MEO2MA-co-OEGMA) gradually shrank into a globular structure from the random coil conformation,
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and became more hydrophobic. Therefore, at this condition, the PEG segments are mainly in the corona of the self-assembled structures, whereas the hydrophobic P(MEO2MA-co-OEGMA) and PCL segments are mainly in the core. The micelles tended to aggregate into compounding micelles or aggregates of larger size (Rh = 103.5 nm, PDI = 0.108). When DTT was added to the micelles solution at 25 °C, the disulfide bonds linking the P(MEO2MAco-OEGMA) and PCL blocks were broken in the reaction with DTT. As a result, the hydrophilic P(MEO2MA-co-OEGMA) blocks were shedded from the micelles. The micelles aggregated into aggregates of large size (Scheme 1). The apparent Rh increased to 238.4 nm. Because of the hydrogen bonding interaction between hydrophobic PCL-A and hydrophilic PEG-U, the supramolecular block copolymer micelles were unstable at acidic pH and high salt concentration. In order to evaluate the
Scheme 1. Schematic presentation of the micelle of P(MEO2MA-co-OEGMA)-SS-PCL-A– U–PEG and the multi-stimuli response.
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responsive ability, the micelles were treated with a different pH buffer and salt concentration and the sizes of the micelles were recorded by DLS measurement. As shown in Figure S8 (Supporting Information), in a 0.12 M salt solution at pH 7.4, the apparent Rh of the micelles (62.8 nm) shows a slight increase compared with that in deionized water. When the salt concentration increased to 0.56 M at pH 7.4, the apparent Rh of the micelles increased to 85.4 nm, which indicated that the hydrogen bonding interaction obviously decreased at high salt concentrations. The hydrophilic PEG was detached from the micelles, the hydrophobicity of the micelles increased. Therefore, aggregation of the micelles occurred to some degree. Aggregates with larger size formed (Scheme 1). When the pH was lowered to 5.0 (0.12 M), the apparent Rh of the micelles also increased to 104.1 nm. This can be explained by the increased protonation of the nitrogen atoms of the nucleobases and the subsequent detachment of hydrophilic PEG blocks, which caused the aggregation of micelles to form large aggregates. In order to further understand the thermoresponse of micelles, the change of the apparent Rh at different temperatures was investigated. Figure S9 (Supporting Information) shows the plot of the apparent Rh of the micelles in aqueous solution as a function of temperature. In the lower temperature ranges, the apparent Rh values are relatively small and change slightly. In contrast, the values increase significantly in the higher temperature ranges. For example, the apparent Rh of the micelles was about 59.2 nm at 31 °C, but the apparent Rh increased to about 146.2 nm at 42 °C. At low temperatures, the P(MEO2MAco-OEGMA) chains existed in random coil conformation owing to the hydrogen-bonding interaction between the polymer and the water molecules. When the temperature increased to a critical value, the polymer chains shrank into a globular structure because the hydrogen bonds between the ether oxygen of P(MEO2MA-co-OEGMA) chains and water molecules collapsed and the block became hydrophobic. Therefore, the hydrophilic–hydrophobic balance underwent obvious modifications and aggregation of the micelles occurred. The variable-temperature 1H NMR of P(MEO2MA-co-OEGMA)-SS-PCL-A– U–PEG in D2O is shown in Figure S10 (Supporting Information) and the result further confirmed the thermoresponse of the micelles. The morphologies of the micelles at different conditions are shown in Figure 2. It can be seen from Figure 2a that dispersed spherical micelles can be obtained at 25 °C in aqueous solutions. However, when the temperature increased to 45 °C, larger micelles and aggregates were observed. Similar phenomena can be found in Figure 2c. At acidic pH (5.0) and high salt concentration (0.56 M) conditions, the stability of the micelles decreased and the obvious aggregation occurred. After DTT was added
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Figure 2. TEM images of P(MEO2MA-co-OEGMA)-SS-PCL-A–U– PEG micelles after exposure to various stimuli: a) 25 °C; b) 45 °C; c) pH 5.0 (0.56 M Na+); d) DTT (10 mM) (Concentration: 1 mg mL−1).
into the solution system and stirred for 48 h in Ar atmosphere at 25 °C, the regular micelles could not be observed in Figure 2d, because the disulfide bonds linking the P(MEO2MA-co-OEGMA)block and PCL block was broken after the reaction with DTT. The serious modification of the hydrophilic–hydrophobic balance changed the morphology of the micelles, leading to the aggregation and precipitation of irregular micelles. Therefore, the TEM results further confirmed the thermo, redox, salt, and pHmulti-responsive properties of P(MEO2MA-co-OEGMA)-SSPCL-A–U–PEG micelles. 3.3. Multi-Controlled Release Properties of Supramolecular Micelles The controlled release properties of Nile red were investigated for different temperature, pH, salt concentration, and DTT solutions. As shown in Figure 3, there was a slow release of about 6.2% of the incorporated Nile red within 120 h in deionized water. In a 0.56 M salt at pH 7.4, about 30.4% Nile red was released in 120 h. A high salt concentration led to the dissociation of the hydrogen bonds and the modification of the hydrophilic–hydrophobic balance of the micelles, followed by a comparatively fast release of the loaded Nile red. When the micelles were dispersed in an acidic environment with a pH value of 5.0, however, Nile red molecules entrapped in the supramolecular block copolymer micelles would be quickly released with the breakage of PEG segments from the micelles and the modification of the hydrophilic–hydrophobic balance. In 0.12 M salt solution at pH 5.0, about 41.8% Nile red was released in 120 h. It can be seen that an acidic medium significantly accelerated the release of Nile red from the micelles. The fast release of Nile red from micelles in an
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acidic pH values, and high salt concentrations compared to low temperature, physiological pH, and low salt concentrations. In addition, adding DTT to the micelles solution would rapidly increase the release of Nile red. Compared with other block copolymer systems, this copolymer can conveniently achieve multi-stimuli responses and corresponding variations of the structure of the micelles. It can be envisaged that such a supramolecular block copolymer with a unique structure and thermo, redox, salt, and pH multi-stimuli responses has potential applications in novel smart drug-delivery systems and intelligent nanomaterial fields.
Supporting Information Figure 3. Release profiles of Nile red from P(MEO2MA-co-OEGMA)SS-PCL-A–U–PEG micelles by altering the temperature, pH value, Na+ concentration, and adding DTT.
acidic environment was likely due to the protonation of the amino group of the nucleobases and the shedding of the PEG blocks in the micelles shell in acidic conditions and the serious modification of the hydrophilic–hydrophobic balance of the micelles. Moreover, when the temperature of the micelles solution was heated to 45 °C the release of Nile red was about 37.0% in 120 h, which should be attributed to the hydrophilic P(MEO2MA-co-OEGMA) chains which became hydrophobic at this temperature leading to aggregation and deformation of the micelles. After adding DTT to the micelles solution, the release of Nile red reached about 69.0%. Under the reaction of DTT, lots of hydrophilic P(MEO2MA-co-OEGMA) chains were broken from the micelle shell and the serious modification of the hydrophilic–hydrophobic balance led to the serious deformation, aggregation, and destruction of the micelles, which was beneficial for the release of encapsulated Nile red molecules. As a result, multi-controlled release properties can be achieved by altering the temperature, pH, salt concentration, and adding DTT to the micelle solution.
4. Conclusions A novel type of supramolecular block copolymer, P(MEO2MA-co-OEGMA)-SS-PCL-A–U–PEG, with a thermoresponsive disulfide bond and molecular recognition of nucleobases was synthesized. The supramolecular block copolymer could self-assemble into temperature, redox, pH, and salt multi-responsive micelles with a low CMC in aqueous solution. Micelles of different size and morphologies were obtained by adjusting the hydrophilic–hydrophobic balance via altering the temperature, pH value, salt concentration, and adding DTT. These micelles were found to release Nile red more rapidly at high temperature,
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Supporting Information is available from the Wiley Online Library or from the author. Acknowledgements: The authors thank the financial support of the National Key Technology R&D Program (No. 2012BAI15B061), the National Basic Research Program of China (973 Program: 2011CB013805), and the National High Technology Research and Development Program of China (No. 2013AA032202). Received: May 30, 2014; Revised: July 5, 2014; Published online: September 5, 2014; DOI: 10.1002/marc.201400308 Keywords: micelles; self-assembly; stimuli-sensitive polymers; supramolecular structures
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