Amphiphilicity
Tuning Polymeric Amphiphilicity via Se–N Interactions: Towards One-Step Double Emulsion for Highly Selective Enzyme Mimics Xiaopeng Huang, Ruochen Fang, Dingguan Wang, Jian Wang, Huaping Xu,* Yapei Wang,* and Xi Zhang The W/O/W double emulsion, an artful microstructure available to encapsulate two immiscible compounds together, is of particular significance in the areas of food industry, cosmetic, drug vehicle, agricultural chemicals, industrial production.[1] It can also be exploited as the template for fabrication of hollow, porous and anisotropic microparticles, opening up a fascinating top-down approach for the micro/nano- technology.[2] Conventional approaches to double emulsion usually involve a two-step emulsification process as well as two distinctive surfactants—hydrophilic and hydrophobic emulsifiers. Though extensively used, this process exhibits three inherent weaknesses: 1) relatively low production efficiency in terms of time and equipment cost; 2) adverse effects from the surfactants including poor biocompatibility and masking effects in subsequent applications; and 3) possible instability of the primary emulsion during the re-emulsification.[3] Therefore, the one-step double emulsion is drawing increasing attention as an alternative approach. Previous studies on one-step methods can be generally divided into two pathways. One is through the precise tailoring of the hydrophobic and hydrophilic combinations consisting of surfactants[4] or surface-active materials[5] to a suitable amphiphilicity, together with elaborate control over the emulsification process. It provides a feasible platform, yet still does not remove the problems with unwanted emulsifiers. The other is definitely attractive because it only uses one single-component synthetic amphiphilic copolymer to stabilize both the inner W/O and outer O/W interfaces. Yet an appropriate amphiphile for the formation of one-step double emulsions is usually uncertain and a certain number of polymerization trials are required.[6a-c] Easier approaches X. Huang, D. Wang, J. Wang, Prof. Y. Wang Department of Chemistry Renmin University of China Beijing 100872, China E-mail: [email protected] R. Fang, Prof. H. Xu, Prof. X. Zhang Key Lab of Organic Optoelectronics and Molecular Engineering Department of Chemistry Tsinghua University Beijing 100084, China E-mail: [email protected] DOI: 10.1002/smll.201402271 small 2014, DOI: 10.1002/smll.201402271
are to utilize stimuli responsive polymers, the amphiphilicity of which can be altered under triggers like temperature or pH value.[6d,e] However, an accessible way that can easily tune the polymer amphiphilicity and simultaneously impart specific function groups to the emulsion system has not yet been reported. Tuning the polymeric amphiphilicity has been extensively studied in the field of self-assembly, providing various routes to control the amphiphilic behaviors ranging from the molecular level to the nanoscale.[7] Of these, the Se-N dynamic covalent bond has been recently reported to produce selfassembling reverse micelles in organic solvent, which may provide an available tool to control the amphiphilic property of materials.[8] Inspired by this preliminary work, we here propose a facile synthetic protocol to acquire one-step double emulsions without any additional surfactants. Selenium-containing small molecules, as a versatile additive, can gradually tune the amphiphilicity of the block copolymer, beneficial for the predictable phase inversion of the emulsion system. The alteration of the polymer amphiphilicity via the selenium-containing small molecule is shown in Figure 1. Pure block copolymer with a pyridine group, PS-b-P4VP (Mw ≈ 8800 g/mol, PDI = 1.15), is hydrophobic and gives a water contact angle of more than 90° (Figure 1b, Table S1). After gradually reacting with phenylselenyl bromide (PhSeBr), a substantial decrease in the water contact angles is observed, suggesting the formation of amphiphilic polymer-small molecule mixtures (Figure 1c–e, Table S1). Evidence is also provided from a stepwise enhancement of the polar component of surface energy, accompanied by a decrease in the dispersive component. This confirms the controllable adjustment of the polymer-water affinity. In addition, these amphiphilic mixtures self-assembled to reverse micelles in dichloromethane (DCM), displaying an increasing Z-average mean size with the enhancement of polymer amphiphilicity, whereas the pure copolymer cannot give any valid measures (Figure 1g). This amphiphilicity alteration, according to the discussed work,[8] is because of the formation of Se-N covalent bond, which would further generate the N-Se cation and the Br counter ion and hence impart the hydrophilic property to the P4VP segment, as schematically depicted in Figure 1a. The quaternization of the P4VP segment by the PhSeBr can be seen from FTIR spectra
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as emulsion 1–0.2, Figure 2b, e), relatively hydrophobic structure prevents the access of external water. With a higher water volume, the emulsion systme is directed by the Ostwald arrangement theory and induces a catastrophic phase inversion, ultimately yielding the O/W abnormal emulsion. When the copolymer amphiphilicity is further increased (designated as emulsion 1–0.5, Figure 2c, f), the amphiphilic structure has a tendency to attract and stabilize the external water. Therefore, an internal W/O interface and an external O/W interface are formed to obey the Bancroft rule and the Ostwald arrangement theory respectively. We thus acquired W/O/W double emulsions with one or several internal water droplets. Similarly, mixture with a higher amphiphilic property produced more remarkable W/O/W double emulsion with every oil globule containing a large number of water droplets (designated as emulsion 1–1, Figure 2d, g). The catastrophic phase separation behaviors are further explored from the Figure 1. (a) The schematic of tuning the amphiphilicity of PS-b-P4VP via Se-N interactions. perspective of water-oil ratio. As illus(b-e) Water contact angles of PS-b-P4VP interacting with PhSeBr at different molar ratios trated in Table 1 and Figure S3, W/O between the pyridine group and PhSeBr of (b) 1:0; (c) 1:0.2; (d) 1:0.5; and (e) 1:1. (f) Surface normal emulsions appear in all the emulenergy and its dispersive and polar components, of the pure PS-b-P4VP and amphiphilic sion systems with different amphiphilic mixtures. (g) DLS Z-average mean size of amphiphilic aggregates in dichloromethane. mixtures when the water-oil ratio is 1:5. When the water volume is equivalent to (Figure S1), in which the characteristic peaks corresponding the oil phase, catastrophic phase inversions emerges, inversing to the C = N stretching vibration of the pyridine ring at the original W/O normal emulsions into O/W abnormal 1557 cm−1 and 1415 cm−1 disappear, and a new characteristic emulsions. Emulsion 1–0.2 and emulsion 1–0.5 show the peak at 1637 cm−1 forms and gradually strengthens. coexistence of the W/O emulsions and O/W emulsions, while We thereafter emulsified these polymer organic solu- emulsion 1–1 completely exhibits O/W abnormal emulsions. tions in water (the water-oil ratio is 5:1) via high-speed This means amphiphilic structure with larger hydrophilic homogenization without the addition of any surfactants. As portion is more advantageous for the phase inversion of expected, the hydrophobic PS-b-P4VP produced a clear seg- the emulsion. Also, this remarkable amphiphilic property, regation of water and oil phases immediately after homog- as described above, would create W/O/W double emulsion enization (Figure S2). In contrast, the amphiphilic mixtures when the water volume is excessive. In short, through elabogave various types of emulsions after one-step emulsifica- rately regulating the phase inversion behaviors in emulsion tion (Figure 2b–d). It should be noted that these emulsions system, W/O/W double emulsions can be obtained via oneare not exactly the same as the normal W/O or O/W single step homogenization. In contrast to reported abnormal emulsions that show emulsions. Phase inversion theory can account for these behaviors. According to the Bancroft rule, these amphi- short-term stability, our emulsions are stable for more than philic mixtures, preferentially dissolved in oil phase (DCM) one month. Apart from the steric repulsion derived from and further self-assembled to reverse micelles, are in favor the long-chain copolymer structure,[6a,10] the electrostatic of the formation W/O simple emulsion. Another theory, the Ostwald arrangement concept, elucidates that the emulsion Table 1. The resulted emulsion types under different water-oil ratio system is more inclined to the stable O/W simple emulsion (horizontal) and polymer-small molecule mixtures with different when the water volume is high enough and reaches a critical amphiphilic degree (vertical). W:O = 1:5 W:O = 1:1 W:O = 5:1 value. Phase inversion between the water and oil phases will occur when the most suitable emulsion type has to make a Emulsion 1–0.2 W/O W/O + O/W O/W selection between the two theories, resulting in the emer- Emulsion 1–0.5 W/O W/O + O/W W/O/W gence of “abnormal emulsion”.[3,4,6b,9] In our case, when the Emulsion 1–1 W/O O/W W/O/W copolymer amphiphilicity is slightly increased (designated
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small 2014, DOI: 10.1002/smll.201402271
Tuning Polymeric Amphiphilicity via Se–N Interactions
( a)
One-step emusification
(b)
(c)
(d)
(e) 1:0.2
(f) 1:0.5
(g)
o/w
w/o/w
1:1
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for the largely amphiphilic mixtures. This implies that, templated from the one-step emulsion, we can obtain intriguing porous particles with high specific surface area, hopeful to be high-performance materials in practice. In light of the introduction of seleniumcontaining small molecular substance, our designed selenium-containing particles are anticipated to play the role of remarkable glutathione peroxidase (GPx) mimics. Selenium-containing active center is the kernel ingredient of GPx, an extremely important mammalian antioxidant enzyme that can catalyze the reduction of superfluous harmful hydroperoxides (ROOH) to avirulent hydroxyl compounds (ROH) based on the glutathione (GSH) substrate (Scheme S1).[11] On the basis of this concept, the catalytic activity of these enzyme candidates was measured according to the typical Hilvert method, in which 3-carboxy-4-nitrobenzenethiol (TNB, Scheme S2) was taken as the glutathione alternative. To be specific, the GPx-catalyzed reaction can be described as follow, [12] GPx mimic
Figure 2. (a) Schematic of the one-step emulsification. The amphiphilic blends are first dissolved in dichloromethane and the water-dichloromethane ratio is 5:1. (b-g) Optical micrographs and schematic of various emulsions prepared by different amphiphilic blends with the molar ratios between the pyridine group in the PS-b-P4VP and PhSeBr from 1:0.2 (b, e), 1:0.5(c, f) to 1:1(d, g).
2TNB + ROOH ⎯⎯⎯⎯⎯ → DTNB + ROH + H 2O
(1)
An appreciably high-efficient catalytic activity was observed among all the three sorts of the GPX mimics with regard to two distinctive hydroperoxides, hydrophilic hydrogen peroxide (H2O2) and hydrophobic cumenehydroperoxide (CUOOH, Scheme S2), as illustrated in Figure 4a, b
repulsion caused by the quaternization of the P4VP segment also plays a significant role for the long-term stabilization. In addition, we also used the conductivity measurement to characterize the gradually (b) (c) (a) enhanced quaternization degree of the P4VP segment based on the as-prepared emulsion. Pure PS-b-P4VP, failing to form an emulsion, gives a low conductivity of 2.49 mS/cm, whereas emulsions stabilized by amphiphilic mixtures show a proportional relationship with the quaternization 5µm 5µm 5µm degree of PS-b-P4VP (Figure S4). For further applications, we are more (f) (d) (e) concerned if these structures can remain rather than coalesce or aggregate after the solvent evaporation, since it can provide more stable microstructure and get rid of the solvent toxicity. Figure 3 clearly indicates the received particles after rapid solvent evaporation. Solid particles 5 µm 5 µm 5 µm 1µm with an average size of 1 µm were collected from the O/W single emulsion. The Figure 3. Optical micrographs and SEM graphs of the particles obtained through the rapid W/O/W abnormal emulsion, however, solvent evaporation of the corresponding emulsions. The oil phase contains different gave hollow particles for the moderately amphiphilic blends with the molar ratios between the pyridine group in the PS-b-P4VP and amphiphilic mixtures and porous particles PhSeBr from 1:0.2 (a, d), 1:0.5 (b, e) to 1:1 (c, f). small 2014, DOI: 10.1002/smll.201402271
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respectively, confirming that this porous particle mimic has a higher affinity to the hydrophobic substrate. In conclusion, through controllably tuning the polymeric amphiphilicity by virtue of small molecule PhSeBr, we have successfully acquired a line of desired emulsions via one-step homogenization. Abnormal emulsions, induced by phase inversion behaviors, are regulated using different amphiphilic mixtures under different water-oil ratios. Particularly the one-step double emulsion can be produced in an easy, efficient and surfactantfree way. Moreover, porous microparticles with high specific area can be obtained on the strength of this double emulsion template, displaying the exceptional function of being highly selective artificial GPx enzyme mimics. Selenium-containing small molecule, in this design, plays the versatile roles of both the “amphiphicity adjuster” and the “function donator”. Based on the Figure 4. (a, b) Catalytic activity plots of different particle mimics. The selenium concentration of these particles is 30 µM. The assays are performed with TNB (600 µM) with hydrophilic covalent or supramolecular interactions, a H2O2 (a) or hydrophobic CUOOH (b). (c, d) The best-fit Lineweaver-Burk plots of the porous large number of small molecules with specific functional groups can be employed particle mimics in the substrate of TNB with H2O2 (c) or CUOOH (d). to tune the amphiphilicity of the polymer towards a one-step multiple emulsion. We and Table S2, 3. The initial reduction rate of the TNB with anticipate that this methodology will find applications in drug H2O2 is 2.66 µM/min, while the participation of particle delivery, chemo-catalysis, and biosensors. mimics (Particle 1–0.2, 1–0.5, 1–1) effectively raises this value to 5.25 µM/min, 6.84 µM/min, and 7.10 µM/min, respectively. This effect is more substantial in the reaction of TNB with CUOOH, presenting the initial reaction rate from 0.65 µM/ min (without enzyme mimic) to 4.42 µM/min, 5.93 µM/min, Acknowledgements and 7.43 µM/min for the solid, hollow and porous microparticles, respectively. All these results suggest that catalytic This work was financially supported by the National Natural activity is regularly increased with the enhancement of the Science Foundation of China (51373197, 21422407, 21121004), specific surface area due to the porous structure templated the National Basic Research Program of China (2013CB834502), from the double emulsion. Apparently, the designed porous Trans-Century Training Programme Foundation for the Talents by particle mimic (Particle 1–1) possesses the most exceptional the State Education Commission (NCET-12–0530). catalytic activity, exhibiting the maximum catalytic enhancement of 2.67-fold and 11.43-fold for the H2O2 and CUOOH, respectively. Additionally, as the mimic is inherently more compatible with hydrophobic matter (dissolved in DCM [1] a) N. Garti, C. Bisperink, Curr. Opin. Colloid In. 1998, 3, 657; b) H. Okochi, M. Nakano, Adv. Drug Deliv. Rev. 2000, but not water), it may show higher catalytic activity for the 45, 5; c) A. Y. Khan, S. Talegaonkar, Z. Iqbal, F. J. Ahmed, hydrophobic CUOOH than the hydrophilic H2O2. To verify R. K. Khar, Curr. Drug Deliv. 2006, 3, 429; d) S.-H. Hu, this hypothesis, we further investigated the catalytic kinetics, B.-J. Liao, C.-S. Chiang, P.-J. Chen, I.-W. Chen, S.-Y. Chen, determining the relationship between the initial reaction rate Adv. Mater. 2012, 24, 3627; e) H. Z. An, M. E. Helgeson, and the concentration of one substrate, TNB, according to the P. S. Doyle, Adv. Mater. 2012, 24, 3838. Michaelis-Menten model, that is [2] a) M. Windbergs, Y. Zhao, J. A. Heyman, D. A. Weitz, J. Am.
Vmax [TNB] V= K m + [TNB]
(2)
By means of Lineweaver-Burk Plot, that is, 1/V versus 1/[TNB], we can thereby obtain the best fitted results (Figure 4c,d). The calculated Michaelis constants (Km) for the H2O2 and CUOOH assay are 597 µM and 332 µM,
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Chem. Soc. 2013, 135, 6649; b) C. H. Chen, R. K. Shah, A. R. Abate, D. A. Weitz, Langmuir 2009, 25, 4320; c) D. Lee, D. A. Weitz, Adv. Mater. 2008, 20, 3498; d) X. Huang, Q. Qian, Y. Wang, Small 2014,10, 1412. [3] a) M. F. Ficheux, L. Bonakdar, F. Leal-Calderon, J. Bibette, Langmuir 1998, 14, 2702; b) J. M. Morais, O. D. Santos, S. E. Friberg, J. Disper. Sci. Technol. 2010, 31, 1019. [4] a) J. M. Morais, O. D. Santos, J. R. Nunes, C. F. Zanatta, P. A. Rocha-Filho, J. Disper. Sci. Technol. 2008, 29, 63;
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small 2014, DOI: 10.1002/smll.201402271
Tuning Polymeric Amphiphilicity via Se–N Interactions
[5]
[6]
[7]
[8]
b) J. M. Morais, P. A. Rocha-Filho, D. J. Burgess, Langmuir 2009, 25, 7954; c) J. M. Morais, P. A. Rocha-Filho, D. J. Burgess, Langmuir 2010, 26, 17874. a) J. Xu, G. Chen, R. Yan, D. Wang, M. Zhang, W. Zhang, P. Sun, Macromolecules 2011, 44, 3730; b) H. Wang, M. Wang, X. Ge, Polymer 2008, 49, 4974; c) Q. Qian, X. Huang, X. Zhang, Z. Xie, Y. Wang, Angew. Chem. Int. Ed. 2013, 52, 10625. a) J. A. Hanson, C. B. Chang, S. M. Graves, Z. Li, T. G. Mason, T. J. Deming, Nature 2008, 455, 85; b) L. Hong, G. Sun, J. Cai, T. Ngai, Langmuir 2012, 28, 2332; c) M. Pradhan, D. Rousseau, J. Colloid. Interf. Sci. 2012, 386, 398; d) B. P. Binks, R. Murakami, S. P. Armes, S. Fujii, Angew. Chem. Int. Ed. 2005, 44, 4795; e) L. Besnard, F. Marchal, J. F. Paredes, J. Daillant, N. Pantoustier, P. Perrin, P. Guenoun, Adv. Mater. 2013, 25, 2844. a) Y. Wang, H. Xu, X. Zhang, Adv. Mater. 2009, 21, 2849; b) Y. Zhou, W. Huang, J. Liu, X. Zhu, D. Yan, Adv. Mater. 2010, 22, 4567; c) G. Chen, M. Jiang, Chem. Soc. Rev. 2011, 40, 2254; d) Q. Duan, Y. Cao, Y. Li, X. Hu, T. Xiao, C. Lin, Y. Pan, L. Wang, J. Am. Chem. Soc. 2013, 135, 10542. Y. Yi, H. Xu, L. Wang, W. Cao, X. Zhang, Chem. Eur. J. 2013, 19, 9506.
small 2014, DOI: 10.1002/smll.201402271
[9] a) J. L. Salager, L. Marquez, A. A. Pena, M. J. Rondon, F. Silva, E. Tyrode, Ind. Eng. Chem. Res. 2000, 39, 2665; b) E. Tyrode, I. Mira, N. Zambrano, L. Márquez, M. Rondon-Gonzalez, J. L. Salager, Ind. Eng. Chem. Res. 2003, 42, 4311; c) G. Sun, Z. Li, T. Ngai, Angew. Chem. Int. Ed. 2010, 49, 2163. [10] N. Garti, A. Aserin, Adv. Colloid Interface Sci. 1996, 65, 37. [11] a) H. Xu, W. Cao, X. Zhang, Acc. Chem. Res. 2013, 46, 1647; b) G. Mugesh, W. W. du Mont, H. Sies, Chem. Rev. 2001, 101, 2125; c) X. Zhang, H. Xu, Z. Dong, Y. Wang, J. Liu, J. Shen, J. Am. Chem. Soc. 2004, 126, 10556; d) X. Huang, X. Liu, Q. Luo, J. Liu, J. Shen, Chem. Soc. Rev. 2011, 40, 1171. [12] a) I. M. Bell, D. Hilvert, Biochemistry 1993, 32, 13969; b) Y. Wang, H. Xu, N. Ma, Z. Wang, X. Zhang, J. Liu, J. Shen, Langmuir 2006, 22, 5552; c) H. Xu, J. Gao, Y. Wang, Z. Wang, M. Smet, W. Dehaen, X. Zhang, Chem. Commun. 2006, 7,796; d) Y. Tang, L. Zhou, J. Li, Q. Luo, X. Huang, P. Wu, Y. Wang, J. Xu, J. Shen, J. Liu, Angew. Chem. Int. Ed. 2010, 49, 3920. Received: July 30, 2014 Revised: September 21, 2014 Published online:
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Tuning Polymeric Amphiphilicity via Se–N Interactions: Towards One-Step Double Emulsion for Highly Selective Enzyme Mimics Xiaopeng Huang, Ruochen Fang, Dingguan Wang, Jian Wang, Huaping Xu,* Yapei Wang,* and Xi Zhang The W/O/W double emulsion, an artful microstructure available to encapsulate two immiscible compounds together, is of particular significance in the areas of food industry, cosmetic, drug vehicle, agricultural chemicals, industrial production.[1] It can also be exploited as the template for fabrication of hollow, porous and anisotropic microparticles, opening up a fascinating top-down approach for the micro/nano- technology.[2] Conventional approaches to double emulsion usually involve a two-step emulsification process as well as two distinctive surfactants—hydrophilic and hydrophobic emulsifiers. Though extensively used, this process exhibits three inherent weaknesses: 1) relatively low production efficiency in terms of time and equipment cost; 2) adverse effects from the surfactants including poor biocompatibility and masking effects in subsequent applications; and 3) possible instability of the primary emulsion during the re-emulsification.[3] Therefore, the one-step double emulsion is drawing increasing attention as an alternative approach. Previous studies on one-step methods can be generally divided into two pathways. One is through the precise tailoring of the hydrophobic and hydrophilic combinations consisting of surfactants[4] or surface-active materials[5] to a suitable amphiphilicity, together with elaborate control over the emulsification process. It provides a feasible platform, yet still does not remove the problems with unwanted emulsifiers. The other is definitely attractive because it only uses one single-component synthetic amphiphilic copolymer to stabilize both the inner W/O and outer O/W interfaces. Yet an appropriate amphiphile for the formation of one-step double emulsions is usually uncertain and a certain number of polymerization trials are required.[6a-c] Easier approaches X. Huang, D. Wang, J. Wang, Prof. Y. Wang Department of Chemistry Renmin University of China Beijing 100872, China E-mail: [email protected] R. Fang, Prof. H. Xu, Prof. X. Zhang Key Lab of Organic Optoelectronics and Molecular Engineering Department of Chemistry Tsinghua University Beijing 100084, China E-mail: [email protected] DOI: 10.1002/smll.201402271 small 2014, DOI: 10.1002/smll.201402271
are to utilize stimuli responsive polymers, the amphiphilicity of which can be altered under triggers like temperature or pH value.[6d,e] However, an accessible way that can easily tune the polymer amphiphilicity and simultaneously impart specific function groups to the emulsion system has not yet been reported. Tuning the polymeric amphiphilicity has been extensively studied in the field of self-assembly, providing various routes to control the amphiphilic behaviors ranging from the molecular level to the nanoscale.[7] Of these, the Se-N dynamic covalent bond has been recently reported to produce selfassembling reverse micelles in organic solvent, which may provide an available tool to control the amphiphilic property of materials.[8] Inspired by this preliminary work, we here propose a facile synthetic protocol to acquire one-step double emulsions without any additional surfactants. Selenium-containing small molecules, as a versatile additive, can gradually tune the amphiphilicity of the block copolymer, beneficial for the predictable phase inversion of the emulsion system. The alteration of the polymer amphiphilicity via the selenium-containing small molecule is shown in Figure 1. Pure block copolymer with a pyridine group, PS-b-P4VP (Mw ≈ 8800 g/mol, PDI = 1.15), is hydrophobic and gives a water contact angle of more than 90° (Figure 1b, Table S1). After gradually reacting with phenylselenyl bromide (PhSeBr), a substantial decrease in the water contact angles is observed, suggesting the formation of amphiphilic polymer-small molecule mixtures (Figure 1c–e, Table S1). Evidence is also provided from a stepwise enhancement of the polar component of surface energy, accompanied by a decrease in the dispersive component. This confirms the controllable adjustment of the polymer-water affinity. In addition, these amphiphilic mixtures self-assembled to reverse micelles in dichloromethane (DCM), displaying an increasing Z-average mean size with the enhancement of polymer amphiphilicity, whereas the pure copolymer cannot give any valid measures (Figure 1g). This amphiphilicity alteration, according to the discussed work,[8] is because of the formation of Se-N covalent bond, which would further generate the N-Se cation and the Br counter ion and hence impart the hydrophilic property to the P4VP segment, as schematically depicted in Figure 1a. The quaternization of the P4VP segment by the PhSeBr can be seen from FTIR spectra
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as emulsion 1–0.2, Figure 2b, e), relatively hydrophobic structure prevents the access of external water. With a higher water volume, the emulsion systme is directed by the Ostwald arrangement theory and induces a catastrophic phase inversion, ultimately yielding the O/W abnormal emulsion. When the copolymer amphiphilicity is further increased (designated as emulsion 1–0.5, Figure 2c, f), the amphiphilic structure has a tendency to attract and stabilize the external water. Therefore, an internal W/O interface and an external O/W interface are formed to obey the Bancroft rule and the Ostwald arrangement theory respectively. We thus acquired W/O/W double emulsions with one or several internal water droplets. Similarly, mixture with a higher amphiphilic property produced more remarkable W/O/W double emulsion with every oil globule containing a large number of water droplets (designated as emulsion 1–1, Figure 2d, g). The catastrophic phase separation behaviors are further explored from the Figure 1. (a) The schematic of tuning the amphiphilicity of PS-b-P4VP via Se-N interactions. perspective of water-oil ratio. As illus(b-e) Water contact angles of PS-b-P4VP interacting with PhSeBr at different molar ratios trated in Table 1 and Figure S3, W/O between the pyridine group and PhSeBr of (b) 1:0; (c) 1:0.2; (d) 1:0.5; and (e) 1:1. (f) Surface normal emulsions appear in all the emulenergy and its dispersive and polar components, of the pure PS-b-P4VP and amphiphilic sion systems with different amphiphilic mixtures. (g) DLS Z-average mean size of amphiphilic aggregates in dichloromethane. mixtures when the water-oil ratio is 1:5. When the water volume is equivalent to (Figure S1), in which the characteristic peaks corresponding the oil phase, catastrophic phase inversions emerges, inversing to the C = N stretching vibration of the pyridine ring at the original W/O normal emulsions into O/W abnormal 1557 cm−1 and 1415 cm−1 disappear, and a new characteristic emulsions. Emulsion 1–0.2 and emulsion 1–0.5 show the peak at 1637 cm−1 forms and gradually strengthens. coexistence of the W/O emulsions and O/W emulsions, while We thereafter emulsified these polymer organic solu- emulsion 1–1 completely exhibits O/W abnormal emulsions. tions in water (the water-oil ratio is 5:1) via high-speed This means amphiphilic structure with larger hydrophilic homogenization without the addition of any surfactants. As portion is more advantageous for the phase inversion of expected, the hydrophobic PS-b-P4VP produced a clear seg- the emulsion. Also, this remarkable amphiphilic property, regation of water and oil phases immediately after homog- as described above, would create W/O/W double emulsion enization (Figure S2). In contrast, the amphiphilic mixtures when the water volume is excessive. In short, through elabogave various types of emulsions after one-step emulsifica- rately regulating the phase inversion behaviors in emulsion tion (Figure 2b–d). It should be noted that these emulsions system, W/O/W double emulsions can be obtained via oneare not exactly the same as the normal W/O or O/W single step homogenization. In contrast to reported abnormal emulsions that show emulsions. Phase inversion theory can account for these behaviors. According to the Bancroft rule, these amphi- short-term stability, our emulsions are stable for more than philic mixtures, preferentially dissolved in oil phase (DCM) one month. Apart from the steric repulsion derived from and further self-assembled to reverse micelles, are in favor the long-chain copolymer structure,[6a,10] the electrostatic of the formation W/O simple emulsion. Another theory, the Ostwald arrangement concept, elucidates that the emulsion Table 1. The resulted emulsion types under different water-oil ratio system is more inclined to the stable O/W simple emulsion (horizontal) and polymer-small molecule mixtures with different when the water volume is high enough and reaches a critical amphiphilic degree (vertical). W:O = 1:5 W:O = 1:1 W:O = 5:1 value. Phase inversion between the water and oil phases will occur when the most suitable emulsion type has to make a Emulsion 1–0.2 W/O W/O + O/W O/W selection between the two theories, resulting in the emer- Emulsion 1–0.5 W/O W/O + O/W W/O/W gence of “abnormal emulsion”.[3,4,6b,9] In our case, when the Emulsion 1–1 W/O O/W W/O/W copolymer amphiphilicity is slightly increased (designated
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small 2014, DOI: 10.1002/smll.201402271
Tuning Polymeric Amphiphilicity via Se–N Interactions
( a)
One-step emusification
(b)
(c)
(d)
(e) 1:0.2
(f) 1:0.5
(g)
o/w
w/o/w
1:1
w/o/w
for the largely amphiphilic mixtures. This implies that, templated from the one-step emulsion, we can obtain intriguing porous particles with high specific surface area, hopeful to be high-performance materials in practice. In light of the introduction of seleniumcontaining small molecular substance, our designed selenium-containing particles are anticipated to play the role of remarkable glutathione peroxidase (GPx) mimics. Selenium-containing active center is the kernel ingredient of GPx, an extremely important mammalian antioxidant enzyme that can catalyze the reduction of superfluous harmful hydroperoxides (ROOH) to avirulent hydroxyl compounds (ROH) based on the glutathione (GSH) substrate (Scheme S1).[11] On the basis of this concept, the catalytic activity of these enzyme candidates was measured according to the typical Hilvert method, in which 3-carboxy-4-nitrobenzenethiol (TNB, Scheme S2) was taken as the glutathione alternative. To be specific, the GPx-catalyzed reaction can be described as follow, [12] GPx mimic
Figure 2. (a) Schematic of the one-step emulsification. The amphiphilic blends are first dissolved in dichloromethane and the water-dichloromethane ratio is 5:1. (b-g) Optical micrographs and schematic of various emulsions prepared by different amphiphilic blends with the molar ratios between the pyridine group in the PS-b-P4VP and PhSeBr from 1:0.2 (b, e), 1:0.5(c, f) to 1:1(d, g).
2TNB + ROOH ⎯⎯⎯⎯⎯ → DTNB + ROH + H 2O
(1)
An appreciably high-efficient catalytic activity was observed among all the three sorts of the GPX mimics with regard to two distinctive hydroperoxides, hydrophilic hydrogen peroxide (H2O2) and hydrophobic cumenehydroperoxide (CUOOH, Scheme S2), as illustrated in Figure 4a, b
repulsion caused by the quaternization of the P4VP segment also plays a significant role for the long-term stabilization. In addition, we also used the conductivity measurement to characterize the gradually (b) (c) (a) enhanced quaternization degree of the P4VP segment based on the as-prepared emulsion. Pure PS-b-P4VP, failing to form an emulsion, gives a low conductivity of 2.49 mS/cm, whereas emulsions stabilized by amphiphilic mixtures show a proportional relationship with the quaternization 5µm 5µm 5µm degree of PS-b-P4VP (Figure S4). For further applications, we are more (f) (d) (e) concerned if these structures can remain rather than coalesce or aggregate after the solvent evaporation, since it can provide more stable microstructure and get rid of the solvent toxicity. Figure 3 clearly indicates the received particles after rapid solvent evaporation. Solid particles 5 µm 5 µm 5 µm 1µm with an average size of 1 µm were collected from the O/W single emulsion. The Figure 3. Optical micrographs and SEM graphs of the particles obtained through the rapid W/O/W abnormal emulsion, however, solvent evaporation of the corresponding emulsions. The oil phase contains different gave hollow particles for the moderately amphiphilic blends with the molar ratios between the pyridine group in the PS-b-P4VP and amphiphilic mixtures and porous particles PhSeBr from 1:0.2 (a, d), 1:0.5 (b, e) to 1:1 (c, f). small 2014, DOI: 10.1002/smll.201402271
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respectively, confirming that this porous particle mimic has a higher affinity to the hydrophobic substrate. In conclusion, through controllably tuning the polymeric amphiphilicity by virtue of small molecule PhSeBr, we have successfully acquired a line of desired emulsions via one-step homogenization. Abnormal emulsions, induced by phase inversion behaviors, are regulated using different amphiphilic mixtures under different water-oil ratios. Particularly the one-step double emulsion can be produced in an easy, efficient and surfactantfree way. Moreover, porous microparticles with high specific area can be obtained on the strength of this double emulsion template, displaying the exceptional function of being highly selective artificial GPx enzyme mimics. Selenium-containing small molecule, in this design, plays the versatile roles of both the “amphiphicity adjuster” and the “function donator”. Based on the Figure 4. (a, b) Catalytic activity plots of different particle mimics. The selenium concentration of these particles is 30 µM. The assays are performed with TNB (600 µM) with hydrophilic covalent or supramolecular interactions, a H2O2 (a) or hydrophobic CUOOH (b). (c, d) The best-fit Lineweaver-Burk plots of the porous large number of small molecules with specific functional groups can be employed particle mimics in the substrate of TNB with H2O2 (c) or CUOOH (d). to tune the amphiphilicity of the polymer towards a one-step multiple emulsion. We and Table S2, 3. The initial reduction rate of the TNB with anticipate that this methodology will find applications in drug H2O2 is 2.66 µM/min, while the participation of particle delivery, chemo-catalysis, and biosensors. mimics (Particle 1–0.2, 1–0.5, 1–1) effectively raises this value to 5.25 µM/min, 6.84 µM/min, and 7.10 µM/min, respectively. This effect is more substantial in the reaction of TNB with CUOOH, presenting the initial reaction rate from 0.65 µM/ min (without enzyme mimic) to 4.42 µM/min, 5.93 µM/min, Acknowledgements and 7.43 µM/min for the solid, hollow and porous microparticles, respectively. All these results suggest that catalytic This work was financially supported by the National Natural activity is regularly increased with the enhancement of the Science Foundation of China (51373197, 21422407, 21121004), specific surface area due to the porous structure templated the National Basic Research Program of China (2013CB834502), from the double emulsion. Apparently, the designed porous Trans-Century Training Programme Foundation for the Talents by particle mimic (Particle 1–1) possesses the most exceptional the State Education Commission (NCET-12–0530). catalytic activity, exhibiting the maximum catalytic enhancement of 2.67-fold and 11.43-fold for the H2O2 and CUOOH, respectively. Additionally, as the mimic is inherently more compatible with hydrophobic matter (dissolved in DCM [1] a) N. Garti, C. Bisperink, Curr. Opin. Colloid In. 1998, 3, 657; b) H. Okochi, M. Nakano, Adv. Drug Deliv. Rev. 2000, but not water), it may show higher catalytic activity for the 45, 5; c) A. Y. Khan, S. Talegaonkar, Z. Iqbal, F. J. Ahmed, hydrophobic CUOOH than the hydrophilic H2O2. To verify R. K. Khar, Curr. Drug Deliv. 2006, 3, 429; d) S.-H. Hu, this hypothesis, we further investigated the catalytic kinetics, B.-J. Liao, C.-S. Chiang, P.-J. Chen, I.-W. Chen, S.-Y. Chen, determining the relationship between the initial reaction rate Adv. Mater. 2012, 24, 3627; e) H. Z. An, M. E. Helgeson, and the concentration of one substrate, TNB, according to the P. S. Doyle, Adv. Mater. 2012, 24, 3838. Michaelis-Menten model, that is [2] a) M. Windbergs, Y. Zhao, J. A. Heyman, D. A. Weitz, J. Am.
Vmax [TNB] V= K m + [TNB]
(2)
By means of Lineweaver-Burk Plot, that is, 1/V versus 1/[TNB], we can thereby obtain the best fitted results (Figure 4c,d). The calculated Michaelis constants (Km) for the H2O2 and CUOOH assay are 597 µM and 332 µM,
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Chem. Soc. 2013, 135, 6649; b) C. H. Chen, R. K. Shah, A. R. Abate, D. A. Weitz, Langmuir 2009, 25, 4320; c) D. Lee, D. A. Weitz, Adv. Mater. 2008, 20, 3498; d) X. Huang, Q. Qian, Y. Wang, Small 2014,10, 1412. [3] a) M. F. Ficheux, L. Bonakdar, F. Leal-Calderon, J. Bibette, Langmuir 1998, 14, 2702; b) J. M. Morais, O. D. Santos, S. E. Friberg, J. Disper. Sci. Technol. 2010, 31, 1019. [4] a) J. M. Morais, O. D. Santos, J. R. Nunes, C. F. Zanatta, P. A. Rocha-Filho, J. Disper. Sci. Technol. 2008, 29, 63;
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Tuning Polymeric Amphiphilicity via Se–N Interactions
[5]
[6]
[7]
[8]
b) J. M. Morais, P. A. Rocha-Filho, D. J. Burgess, Langmuir 2009, 25, 7954; c) J. M. Morais, P. A. Rocha-Filho, D. J. Burgess, Langmuir 2010, 26, 17874. a) J. Xu, G. Chen, R. Yan, D. Wang, M. Zhang, W. Zhang, P. Sun, Macromolecules 2011, 44, 3730; b) H. Wang, M. Wang, X. Ge, Polymer 2008, 49, 4974; c) Q. Qian, X. Huang, X. Zhang, Z. Xie, Y. Wang, Angew. Chem. Int. Ed. 2013, 52, 10625. a) J. A. Hanson, C. B. Chang, S. M. Graves, Z. Li, T. G. Mason, T. J. Deming, Nature 2008, 455, 85; b) L. Hong, G. Sun, J. Cai, T. Ngai, Langmuir 2012, 28, 2332; c) M. Pradhan, D. Rousseau, J. Colloid. Interf. Sci. 2012, 386, 398; d) B. P. Binks, R. Murakami, S. P. Armes, S. Fujii, Angew. Chem. Int. Ed. 2005, 44, 4795; e) L. Besnard, F. Marchal, J. F. Paredes, J. Daillant, N. Pantoustier, P. Perrin, P. Guenoun, Adv. Mater. 2013, 25, 2844. a) Y. Wang, H. Xu, X. Zhang, Adv. Mater. 2009, 21, 2849; b) Y. Zhou, W. Huang, J. Liu, X. Zhu, D. Yan, Adv. Mater. 2010, 22, 4567; c) G. Chen, M. Jiang, Chem. Soc. Rev. 2011, 40, 2254; d) Q. Duan, Y. Cao, Y. Li, X. Hu, T. Xiao, C. Lin, Y. Pan, L. Wang, J. Am. Chem. Soc. 2013, 135, 10542. Y. Yi, H. Xu, L. Wang, W. Cao, X. Zhang, Chem. Eur. J. 2013, 19, 9506.
small 2014, DOI: 10.1002/smll.201402271
[9] a) J. L. Salager, L. Marquez, A. A. Pena, M. J. Rondon, F. Silva, E. Tyrode, Ind. Eng. Chem. Res. 2000, 39, 2665; b) E. Tyrode, I. Mira, N. Zambrano, L. Márquez, M. Rondon-Gonzalez, J. L. Salager, Ind. Eng. Chem. Res. 2003, 42, 4311; c) G. Sun, Z. Li, T. Ngai, Angew. Chem. Int. Ed. 2010, 49, 2163. [10] N. Garti, A. Aserin, Adv. Colloid Interface Sci. 1996, 65, 37. [11] a) H. Xu, W. Cao, X. Zhang, Acc. Chem. Res. 2013, 46, 1647; b) G. Mugesh, W. W. du Mont, H. Sies, Chem. Rev. 2001, 101, 2125; c) X. Zhang, H. Xu, Z. Dong, Y. Wang, J. Liu, J. Shen, J. Am. Chem. Soc. 2004, 126, 10556; d) X. Huang, X. Liu, Q. Luo, J. Liu, J. Shen, Chem. Soc. Rev. 2011, 40, 1171. [12] a) I. M. Bell, D. Hilvert, Biochemistry 1993, 32, 13969; b) Y. Wang, H. Xu, N. Ma, Z. Wang, X. Zhang, J. Liu, J. Shen, Langmuir 2006, 22, 5552; c) H. Xu, J. Gao, Y. Wang, Z. Wang, M. Smet, W. Dehaen, X. Zhang, Chem. Commun. 2006, 7,796; d) Y. Tang, L. Zhou, J. Li, Q. Luo, X. Huang, P. Wu, Y. Wang, J. Xu, J. Shen, J. Liu, Angew. Chem. Int. Ed. 2010, 49, 3920. Received: July 30, 2014 Revised: September 21, 2014 Published online:
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