A Valid Way of Quasi-Quantificationally Controlling the Self-Assembly

Feb 9, 2009 - To mimic nanostructures assembled by biomolecules in organic cells and achieve precise self-assembly of block copolymers, a simple but ...
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Langmuir 2009, 25, 2757-2764

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A Valid Way of Quasi-Quantificationally Controlling the Self-Assembly of Block Copolymers in Confined Space Yan Li, Rujiang Ma, Lizhi Zhao, Qian Tao, De’an Xiong, Yingli An, and Linqi Shi* Key Laboratory of Functional Polymer Materials, Ministry of Education, and Institute of Polymer Chemistry, Nankai UniVersity, Tianjin 300071, China ReceiVed December 3, 2008. ReVised Manuscript ReceiVed January 12, 2009 To mimic nanostructures assembled by biomolecules in organic cells and achieve precise self-assembly of block copolymers, a simple but valid way is introduced to quasi-quantificationally control the aggregation numbers (Nagg) of polymeric micelles. A three-dimensional and closed microconfinement similar to a cell is constructed by W/O inverse emulsion as the spot for self-assembly of the pH-responsive block copolymer poly(ethylene glycol)-blockpoly(4-vinylpyridine) (PEG-b-P4VP). The Nagg values of the resulting polymeric micelles are effectively controlled by tuning the number of polymer chains encapsulated in isolated water pools. Micelles with different Nagg values are successfully prepared and characterized by atomic force microscopy, transmission electron microscopy, and dynamic light scattering. When the number of polymer chains enclosed in a water pool (Nchain) is less than the average Nagg of normal micelles generated in bulk aqueous solution, the resultant aggregates formed in the confined spaces always have lower Nagg as well as smaller sizes than the normal micelles do, while normal micelles predominantly form when Nchain > Nagg (normal micelle).

Introduction Micellization of amphiphilic block copolymers in block selective solvents has stimulated a great many interests during the past three decades because of their potential applications in various fields, such as materials science, biomimetic chemistry, and drug delivery.1-8 Up until the present, a wide range of morphologies of nanoorganizations, which are based upon selfassembly of di- or triblock copolymers, have been well documented.9-19 Owing to the great resemblance in shapes and structures between polymer aggregates and organic units, study on the morphologies of self-assembled nanoparticles might prove * Corresponding author. E-mail: [email protected]. Telephone: 8622-23506103. Fax: 86-22-23503510. (1) Rosler, A.; Klok, H. A.; Hamley, I. W.; Castelletto, V.; Mykhaylyk, O. O. Biomacromolecules 2003, 4, 859–863. (2) Filali, M.; Meier, M. A. R.; Schubert, U. S.; Gohy, J. F. Langmuir 2005, 21, 7995–8000. (3) Kataoka, K.; Harada, A.; Wakebayashi, D.; Nagasaki, Y. Macromolecules 1999, 32, 6892–6894. (4) Li, Y.; Lokitz, B. S.; Armes, S. P.; McCormick, C. L. Macromolecules 2006, 39, 2726–2728. (5) Meier, M.; Filali, M.; Gohy, J. F.; Schubert, U. S. J. Mater. Chem. 2006, 16, 3001–3006. (6) Yu, S. H.; Colfen, H.; Hartmann, J.; Antonietti, M. AdV. Funct. Mater. 2002, 12, 541–545. (7) Discher, D. E.; Eisenberg, A. Science 2002, 297, 967–973. (8) Licciardi, M.; Giammona, G.; Du, J. Z.; Armes, S. P.; Tang, Y. Q.; Lewis, A. L. Polymer 2006, 47, 2946–2955. (9) Cui, H. G.; Chen, Z. Y.; Zhong, S.; Wooley, K. L.; Pochan, D. J. Science 2007, 317, 647–650. (10) Huang, L. H.; Hu, J.; Lang, L.; Zhuang, X. L.; Chen, X. S.; Wei, Y.; Jing, X. B. Macromol. Rapid Commun. 2008, 29, 1242–1247. (11) Zhang, L.; Eisenberg, A. J. Am. Chem. Soc. 1996, 118, 3168–3181. (12) Mu, M.; Ning, F.; Jiang, M.; Chen, D. Langmuir 2003, 19, 9994–9996. (13) Raez, J.; Tomba, J. P.; Manners, I.; Winnik, M. A. J. Am. Chem. Soc. 2003, 125, 9546–9547. (14) Ma, J. W.; Li, X.; Tang, P.; Yang, Y. J. Phys. Chem. B 2007, 111, 1552– 1558. (15) Geng, Y.; Discher, D. E. J. Am. Chem. Soc. 2005, 127, 12780–12781. (16) Duan, H.; Chen, D.; Jiang, M.; Gan, W.; Li, S.; Wang, M.; Gong, J. J. Am. Chem. Soc. 2001, 123, 12097–12098. (17) Bhargava, P.; Zheng, J. X.; Li, P.; Quirk, R. P.; Harris, F. W.; Cheng, S. Z. D. Macromolecules 2006, 39, 4880–4888. (18) Jiang, Y.; Zhu, J.; Jiang, W.; Liang, H. J. Phys. Chem. B 2005, 109, 21549–21555. (19) Zhu, J.; Jiang, Y.; Liang, H.; Jiang, W. J. Phys. Chem. B 2005, 109, 8619–8625.

fruitful for better understanding the physical principles in the formation of nanostructures existing in organisms. Thus, simulation of bioaggregates with precise structures and peculiar functions becomes a grand challenge up to date. The cell synthesizes and assembles an exact number of biomolecules into aggregates bearing special structures and unique performance. A typical case is the virus, which has a protein coat (capsid) to encase the internal nucleic acid. As a critical part of the virus, the capsid is usually composed of a certain number of capsomeres. A capsomere that can be observed with an electron microscope usually consists of one to six protein subunits (polypeptides). For example, the tobacco mosaic virus (TMV) is one of the most studied viruses whose capsid is formed by 2130 capsomeres with a relative molecular weight of 17500 for each of them. Besides, the capsid of an adenovirus contains 252 capsomeres. In addition to the virus, the monoclonal antibody, another kind of typical bioaggregate, contains much less subunits. The basic structure of this kind of antibody molecule consists of four protein chains and is shaped like a capital letter “Y”. It is obvious that any tiny change in the number of building blocks, which act as the base of various functions of bioaggregates, would lead to variation of the host. Therefore, precise assembly of molecules in number is necessary and crucial for an accurate simulation of bioaggregates. Could man-made molecules mimic those in the cell and also form precise materials? Apparently so.20 Kellermann et al. have provided a convincing demonstration in their work to obtain a near-spherical micelle formed by exactly seven molecules by using amphiphilic dendro-calixarenes.21 However, precise assembly of amphiphilic block copolymers has not been effectively achieved yet, which is a critical problem to be solved in mimicking self-assembly phenomena in organisms. As we all know, conventional self-assembly of amphiphilic block copolymers in block selective solvents occurs spontaneously and the Nagg of the resulting aggregates cannot be controlled quantificationally. A feasible way of preventing spontaneous aggregation of polymer chains and controlling the Nagg of micelles (20) Discher, D. E.; Kamien, R. D. Nature 2004, 430, 519–520. (21) Kellermann, M.; Bauer, W.; Hirsch, A.; Schade, B.; Ludwig, K.; Bottcher, C. Angew. Chem., Int. Ed. 2004, 43, 2959–2962.

10.1021/la803996b CCC: $40.75  2009 American Chemical Society Published on Web 02/09/2009

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precisely is to construct a confined space. Some groups have reported that self-assembly of block copolymers in twodimensional confinement such as nanopores or nanochannels could bring the aggregates some novel structures such as helices, stacked toroids, concentric lamellae, and so on,22-25 which broke through the scale limitation and extended the research content of self-assembly of block copolymers. However, such an open microenvironment could not yet realize a precise control over the Nagg of the aggregates. It is admitted that a lot of biomolecules assemble in the cell, which means the cell could produce various kinds of aggregates. So, a closed and confined microspace similar to the cell should be first constructed. The emulsion technique has, by now, attracted great attention because emulsion droplets could enclose a fixed volume of fluid, which makes it an efficient role for encapsulation and ensures that the internal region of the droplets remain isolated from the outside continuous phase.26-33 As a result, it was born an excellent candidate for confinement, the size of which could be easily tuned by altering the diameter of emulsion droplets. In the present study, a confined microspace is designed via W/O inverse emulsion as the spot of self-assembly of poly(ethylene glycol)-block-poly(4-vinylpyridine) (PEG-b-P4VP). By altering the size of the inverse emulsion droplet and the concentration of the block copolymer solution, the Nagg of micelles formed in water pools can be controlled quasi-quantificationally. Micelles thus obtained show some different characteristics from those formed spontaneously in bulk solution.

Experimental Section Materials. Poly(ethylene glycol) monomethyl ether (CH3OPEG114-OH) (PDI ) 1.05) was purchased from Fluka. 2-Bromo2-methyl-propionyl bromide (BMPB, 98%) and tris(2-aminoethyl)amine (TREN, 96%) were purchased from Acros Organics and used without further purification. Tris-(2-dimethyl aminoethyl) amine (Me6TREN) used as an atom transfer radical polymerization (ATRP) ligand was synthesized according to ref 34. 4-Vinylpyridine (4-VP, Acros Organics, 95%) was dried with CaH2 and distilled under reduced pressure. CuCl was purchased from Aldrich and purified according to ref 35. Span 80 (sorbitan monooleate) was obtained from Sigma-Aldrich and used as received. Triethylamine was purchased from TianJin Chemical Corporation and used after distilling. Other reagents and solvents were of analytical grade and used without further purification. Stir was provided by using a twoblade paddle rod placed in a 25 mL two-neck flask, connected to an overhead stirrer. Synthesis of Diblock Copolymer PEG114-b-P4VP64. The macroinitiator PEG114-Br was synthesized by reacting monomethylended poly(ethylene glycol) with 2-bromo-2-methylpropionyl bromide (BMPB) in toluene with the existence of triethylene amine according to refs 36 and 37. The block copolymer PEG114-b-P4VP64 was synthesized by atom transfer radical polymerization (ATRP) of (22) Yu, B.; Sun, P. C.; Chen, T. H.; Jin, Q. H.; Ding, D. T.; Li, B. H.; Shi, A. C. J. Chem. Phys. 2007, 126. (23) He, X. H.; Song, M.; Liang, H. J.; Pan, C. Y. J. Chem. Phys. 2001, 114, 10510–10513. (24) Li, W.; Wickham, R. A. Macromolecules 2006, 39, 8492–8498. (25) Zhu, Y.; Jiang, W. Macromolecules 2007, 40, 2872–2881. (26) Rocca, S.; Garcia-Celma, M. J.; Caldero, G.; Pons, R.; Solans, C.; Stebe, M. J. Langmuir 1998, 14, 6840–6845. (27) Landfester, K.; Willert, M.; Antonietti, M. Macromolecules 2000, 33, 2370–2376. (28) Hirai, T.; Hodono, M.; Komasawa, I. Langmuir 2000, 16, 955–960. (29) Pautot, S.; Frisken, B. J.; Weitz, D. A. Langmuir 2003, 19, 2870–2879. (30) Cho, Y. S.; Yi, G. R.; Kim, S. H.; Pine, D. J.; Yang, S. M. Chem. Mater. 2005, 17, 5006–5013. (31) Yuan, X.; Fischer, K.; Schartl, W. Langmuir 2005, 21, 9374–9380. (32) Jung, J.; Lee, I. H.; Lee, E.; Park, J.; Jon, S. Biomacromolecules 2007, 8, 3401–3407. (33) Cheng, J.; Chen, J. F.; Wen, L. X. Ind. Eng. Chem. Res. 2007, 46, 6259– 6263.

Li et al. 4-vinylpyridine with PEG114-Br as the macroinitiator and CuCl complexed by Me6TREN as the catalyst.38,39 The composition of the block copolymer was determined by using the 1H NMR spectrum in CDCl3 with the PEG block as the inner standard. The molecular weight (Mn) was determined to be about 1.18 × 104. The polydispersity index (Mw/Mn) of the block copolymer was characterized to be 1.20 by gel permeation chromatography (GPC) with N,Ndimethylformamide (DMF) as the eluent and narrowly distributed poly(methyl methacrylate) as the calibration standard. Preparation of the PEG114-b-P4VP64 Micelle in Bulk Aqueous Solution. A certain amount of PEG114-b-P4VP64 was added into acidic water to give the polymer solution the concentration of 2.5 mg/mL and a pH value lower than the pKa (4.5-4.7) of the P4VP block.38 About 24 h later, freshly distilled triethylamine was added dropwise into the polymer solution under stirring until the pH value just exceeded the pKa of P4VP when PEG-b-P4VP micelles (defined as normal micelles in this Article) were obtained with P4VP as the core and PEG as the shell. The concentration remained almost the same. The micelle solution was stirred for 24 h before characterization. Preparation of Polymer-Containing Emulsions and Micellization of Block Copolymer in Emulsion Droplets. A commonly used nonionic surfactant sorbitan monooleate (Span 80) was employed, since it favors a W/O emulsion due to its marked lipophilic character (HLB ) 4.3). Polymer solutions of different concentrations (0.5, 2.5, and 10 mg/mL) were obtained by dissolving certain amounts of block copolymer in acidic water to give final pH values lower than the pKa of P4VP. Span 80 was added into cyclohexane (0.44/ 10, m/m) and dissolved absolutely under ultrasonic. First, 2 mL of polymer solution of different concentration was added into 10 g of oil solution, and the mixture was stabilized for about 48 h under gentle stirring (about 500 rpm) at ambient temperature. To complete micellization, 30 µL of freshly distilled thiethylamine was injected into the emulsion system; the emulsion was stirred for a further 48 h (velocity keep constant for all experiments). For a huge emulsion, 5.11 g of Span 80/cyclohexane solution (0.11/5, m/m) was used as the oil continuous phase, to which 4.6 mL of aqueous polymer solution with a concentration at 0.014 mg/mL was added and the mixture was stirred for about 48 h before characterization. A similar micellization process was conducted as described above. For the sake of a clear reflection of the figures of droplets through laser scanning confocal microscopy (LSCM), a trace amount of pyranine (trisodium 8-hydroxypyrene-1,3,6-trisulfonate) was introduced into the polymer solution priorly as a fluorescent probe. Breakage of Emulsions. Two approaches were used to break emulsions: (i) acetone was added into emulsions where micellization of block copolymers was completed before measurement and (ii) freeze/thaw-induced demulsification as a effective way of destroying the emulsions was adopted according to ref 40. For the second method, the resultant destroyed emulsion consisted of two layers. The surfactant-rich cyclohexane solution was located in the top layer while the aqueous solution of micelles in the sublayer was then extracted using an injector, and diluted with aqueous solution of the same pH for further characterizations. Light Scattering Measurements. Dynamic light scattering (DLS) was used to determine the sizes of the emulsion droplets and the micelles obtained after demulsification. In this study, DLS measurements were performed at 636 nm by using a laser light scattering spectrometer (BI-200SM) equipped with a digital correlator (BI9000AT). Freshly prepared emulsions were determined directly after being diluted to some degree. Filtering sublayer micelle solutions (34) Ciampolini, M.; Nardi, N. Inorg. Chem. 1966, 5, 41–44. (35) Keller, R. N.; Wycoff, H. D. Inorg. Synth. 1946, 2, 1. (36) Liu, S.; Weaver, J. V. M.; Save, M.; Armes, S. P. Langmuir 2002, 18, 8350–8357. (37) Zhang, W.; Shi, L.; Gao, L.; An, Y.; Li, G.; Wu, K.; Liu, Z. Macromolecules 2005, 38, 899–903. (38) Sidorov, S. N.; Bronstein, L. M.; Kabachii, Y. A.; Valetsky, P. M.; Soo, P. L.; Maysinger, D.; Eisenberg, A. Langmuir 2004, 20, 3543–3550. (39) Zhang, W.; Shi, L.; Ma, R.; An, Y.; Xu, Y.; Wu, K. Macromolecules 2005, 38, 8850–8852. (40) Lin, C.; He, G.; Dong, C.; Liu, H.; Xiao, G.; Liu, Y. Langmuir 2008, 24, 5291–5298.

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Figure 1. (a) Hydrodynamic diameter distribution and (b) AFM image of normal PEG114-b-P4VP64 micelles where the polymer concentration was 0.25 mg/mL. DLS measurement was performed at the scattering angle of 90° at ambient temperature.

Figure 2. Schematic illustration of micellization of PEG-b-P4VP in confined spaces constructed by emulsion droplets.

(about 1 mL) through a 0.45 µm Millipore filter into a clean scintillation vial was manipulated to obtain the samples. Characterizations were conducted at ambient temperature. Laser Scanning Confocal Microscopy (LSCM) Measurements. LCSM (Olympus FV1000S-IX81) was used to observe the morphology of the polymer-containing emulsion droplets. Samples for LSCM were prepared by depositing a drop of sample onto a clean slide, on which a cover glass was then placed. The images were obtained using xy-scan, which provided an optical cross section of the droplets under study.

Transmission Electron Microscopy (TEM) Measurements. TEM measurements were performed with a commercial Philips T20ST electron microscope at an acceleration voltage of 200 kV. To prepare the TEM samples, a small drop of the sample solution was deposited onto a carbon-coated copper electron microscopy (EM) grid and dried under room temperature and atmospheric pressure. Atomic Force Microscopy (AFM) Measurements. Samples for AFM imaging were prepared by depositing a drop of sample onto a freshly cleaved mica plate. Excess sample was wicked away with filter paper. And then they were dried under vacuum at room temperature for at least 2 days. AFM imaging was performed in air through a Multimode NanoScope IV system (Veeco, Santa Barbara, CA) operated in tapping mode.

Results and Discussion As a typical pH-responsive diblock copolymer, PEG-bP4VP is suitable to be chosen for our investigation. The micellization of PEG114-b-P4VP64 in bulk aqueous solution was first studied for the purpose of comparison with that in confined space constructed by an emulsion droplet. Figure 1a shows the hydrodynamic diameter distributions of the PEG114-

Figure 3. Hydrodynamic diameter distributions of emulsions with different polymer concentrations, where the oil continuous phase remain constant in all cases. All DLS measurements were performed at the scattering angle of 90° at ambient temperature.

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Figure 4. Laser scanning confocal microscopy images of emulsions with different polymer concentrations: (a) 0.5 mg/mL (E0.5); (b) 2.5 mg/mL (E2.5); and (c) 10 mg/mL (E10).

Figure 5. AFM images of dry emulsion droplets of different polymer-containing emulsions: (a) E0.5; (b) E2.5; and (c) E10.

b-P4VP64 normal micelles. The apparent average hydrodynamic diameter Dhapp, which could be calculated from ∫0∞ f(Dh) Dh dDh, was 70.8 nm. Atomic force microscopy measurement gave images of the morphology of dried PEG114-b-P4VP64 micelles as shown in Figure 1b. In the AFM photograph, micelles with spherical morphologies can be observed, and the diameters were in the range of 60-80 nm, which agreed well with the results obtained by DLS measurement. The apparent weight-average molar mass Mwapp and the average aggregation number Nagg (the number of diblocks associating to form the micelles) were calculated from the Zimm plot (data are not shown) to be 8.14 × 106 and 693, respectively. However, these values would vary in a certain range if different ways to prepare micelle were employed. To the best of our knowledge, Nagg usually ranges from a few dozen to several thousand for many other kinds of polymeric aggregates and cannot be controlled quantificationally.37,41-47 (41) Khougaz, K.; Zhong, X. F.; Eisenberg, A. Macromolecules 1996, 29, 3937–3949. (42) Yusa, S.; Shimada, Y.; Mitsukami, Y.; Yamamoto, T.; Morishima, Y. Macromolecules 2003, 36, 4208–4215. (43) LaRue, I.; Adam, M.; Pitsikalis, M.; Hadjichristidis, N.; Rubinstein, M.; Sheiko, S. S. Macromolecules 2006, 39, 309–314. (44) Hickl, P.; Ballauff, M.; Jada, A. Macromolecules 1996, 29, 4006–4014. (45) Gan, Z.; Jim, T. F.; Li, M.; Yuer, Z.; Wang, S.; Wu, C. Macromolecules 1999, 32, 590–594. (46) Colombani, O.; Ruppel, M.; Burkhardt, M.; Drechsler, M.; Schumacher, M.; Gradzielski, M.; Schweins, R.; Muller, A. H. E. Macromolecules 2007, 40, 4351–4362. (47) Szczubialka, K.; Ishikawa, K.; Morishima, Y. Langmuir 1999, 15, 454– 462.

Table 1. Variation of Nchain with the Polymer Concentrations Cp (mg/mL) 0.5 2.5 10 a

Rhapp (nm) 76.2 88.8 125.6

Nchaina 40-50 370-380 4230-4240

Nchain is equal to Nagg of aggregates formed in a water pool.

It is obvious that PEG114-b-P4VP64 could assemble spontaneously into a spherical micelle in bulk aqueous solution with a rather high value of Nagg. To achieve a relatively accurate control over Nagg, an experimental setup which aimed to create a polymer-containing, isolated, and confined space surrounded by a continuous outer phase is shown schematically in Figure 2. Nonionic surfactant Span 80 is employed to produce a W/O emulsion that could provide a stable space with a relatively uniform size for micellization of the block copolymer. Acidic aqueous solution of PEG114-b-P4VP64 is prepared in the first instance where the polymer chains dissolve freely due to effective protonation of the P4VP block. It is then mixed with an oil solution of Span 80 followed by a few days’ stabilization under gentle stirring. Accompanying the formation of water droplets, block copolymer molecules could be encapsulated into the confined spaces. It is convenient to control the number of molecular chains in a water pool (defined as Nchain in this Article) by tuning the concentration of polymer solution as well as the size of emulsion droplets on account of the macromolecules homogeneously dispersing in aqueous solution. Some Lewis bases, which are soluble both in the oil continuous phase and internal acid aqueous solution, are introduced to change the pH

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Figure 6. AFM images of the micelles captured after demulsifying with acetone: (a) E0.5; (c) E2.5; and (e) E10. TEM images of the micelles captured after demulsifying with acetone: (b) E0.5; (d) E2.5; and (f) E10.

value of the water region. They are capable of getting through the W/O interface built by the surfactant and into the water region to cancel the positive charge of the P4VP unit, which leads to micellization of the block copolymer. A matter worthy of noting is that when a certain amount of polymer chains are encapsulated in the confined space, they become isolated from the outside world. Thus, the Nagg of the resultant micelle (defined as a confined micelle here) formed in a water pool is just equal to the Nchain, and a precise control over Nchain would definitely result in precise self-assembly of the polymer in number. Assuming that block copolymer molecules disperse homogenously in the water region, the Nchain equals to the Nagg of a confined micelle formed in a water pool and can be calculated by the ratio of the total of macromolecules in the original bulk

polymer solution to the number of droplets in the emulsion system. An expression could be deduced as

4 Nchain ) πCpNA(Rhapp)3 ⁄ Mn 3

(1)

where Cp is the weight concentration of polymer solution, NA denotes Avogadro’s number, Mn is the molecular weight of PEG114-b-P4VP64, and Rhapp means the apparent average hydrodynamic radius of the emulsion droplets. Given that NA and Mn remain constant, the value of Nchain only depends on Cp and Rhapp, which means that the average aggregation number of micelles formed in emulsion droplets can be easily tuned by manipulating Cp and Rhapp. In this work, all emulsions came out to be milky. As the water drops came into being during the stirring process,

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Figure 7. AFM of the micelles captured after freeze/thaw-induced demulsification: (a) E0.5; (c) E2.5; and (d) E10. Detailed TEM image of E0.5 (b).

Figure 8. Hydrodynamic diameter distributions of confined micelles obtained through freeze/thaw-induced demulsification from emulsions with different polymer concentrations. All measurements were performed at the scattering angle of 90° at ambient temperature.

surfactant molecules dispersed in oil phase were adsorbed at the interface of water/oil and formed a monolayer that helped to stabilize the emulsion and reduce coalescence of water droplets. However, it required an extended period of time to fully cover the interface; as a result, the emulsion droplets

were much less robust and required a long equilibration time. Simple O/W and W/O emulsions are inherently unstable from a thermodynamic point of view;48 however, these droplets in our work could be stabilized for at least several hours without further fusion if no disturbance was exerted. In contrast, self-

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Figure 9. LSCM image of emulsion droplets of micrometer level. The concentration of polymer solution was 0.014 mg/mL, and pyranine was used as the fluorescent probe.

assembly of block copolymers usually completes in a fairly short time, so all emulsions in our experiment were adequate for their missions. Hydrodynamic diameter distributions of the freshly prepared emulsions are shown in Figure 3. With an increase in concentrations of the polymer solutions, the Rhapp of the emulsion droplets obtained by DLS ranges from 76.2 to 125.5 nm. A possible reason for this tendency lies with the influence of viscosity of the internal phase. In our work, the enhancement of concentrations of aqueous polymer solutions led to an increase in the viscosity of the internal phase. Thus, larger droplets were preferred when the intensity of stirring was kept constant. It was reported that, to improve the stability of the inverse emulsion, some salts and water-soluble polymers were dissolved in the disperse phase to elevate the osmotic pressure against the Laplace pressure.49 It is worthy noting that the improvement of emulsion stability probably has a significant impact on the diameter distribution of the dispersed droplets, and narrowly distributed ones are expected to appear when sufficient polymer is added. It can be observed from Figure 3 that the diameter distributions of emulsions with polymer concentrations at 2.5 mg/mL (denoted as E2.5) and 10 mg/mL (denoted as E10) are remarkably narrower than that of a emulsion with a polymer concentration of 0.5 mg/mL (denoted as E0.5). Figure 4 shows the laser scanning confocal microscopy images of polymer-containing emulsions. Well-defined spherical emulsion droplets are observed in all pictures. There is a considerably narrow size distribution of emulsion droplets in Figure 4b and c along with some slight droplet aggregates, while a broad one can be seen in Figure 4a, which is consistent with the result shown in Figure 3. The monodispersity of droplets determined by DLS and LSCM makes them an eligible confined space for self-assembly of PEG114-b-P4VP64. Table 1 shows the variation of Nchain calculated by eq 1 with the polymer concentrations. For the cases of E0.5 and E2.5, Nchain comes out to be smaller than the Nagg of the normal micelle, which would probably endow these confined micelles with smaller Nagg, whereas the number of polymer chains enclosed in a droplet far exceeds normal micelles’ Nagg for the case of E10, which stimulated our further interest in figuring out the (48) Leal-Calderon, F.; Gerhardi, B.; Espert, A.; Brossard, F.; Alard, V.; Tranchant, J. F.; Stora, T.; Bibette, J. Langmuir 1996, 12, 872–874. (49) Yafei, W.; Tao, Z.; Gang, H. Langmuir 2006, 22, 67–73.

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Figure 10. Hydrodynamic diameter distribution of PEG114-b-P4VP64 micelles formed in huge confined spaces, where the polymer concentration was 0.014 mg/mL. DLS measurements were performed at the scattering angle of 90° at ambient temperature.

status of the polymer aggregates in the water house. The possible conclusion of this issue will be provided in the following text. As has been stated above, spherical normal micelles of PEG114b-P4VP64 in bulk solution with a narrow size distribution have been obtained. Then, the shapes and structures of those aggregates generated in microconfinements are the focus of our following discussion. Figure 5 shows the AFM images of dry polymercontaining emulsion droplets, in which a well-defined and spherical morphology can be clearly seen for all the cases. The diameters of these dry particles are narrowly distributed and range from 30 to 40 nm for E0.5, 40 to 50 nm for E2.5, and 130 to 160 nm for E10. The spherical particles in Figure 5a and b have markedly smaller sizes compared with normal PEG114-bP4VP64 micelles, which indicates the formation of spherical micelles with smaller Nagg in original water cages of E0.5 and E2.5. Though spherical morphology can also be observed in Figure 5c, the diameter appears much larger than that of normal micelles, which probably implies much more polymer chains are located in an emulsion droplet. To visualize the true face of the confined micelles, two alternative approaches were considered in our process to break emulsion. In the first, a volume of acetone (about 5 times larger than the emulsion) was introduced into emulsion systems. When the emulsion systems were destroyed, micelles existing in original confined spaces ran into the current bulk world without any change in their morphologies and configurations. A combination of AFM and TEM was used to visualize the morphologies of the captured micelles (shown in Figure 6). As presented in Figure 6a-d, the true face of typically spherical micelles could be observed, and the diameter scopes are 20-30 nm for the micelles with a Nagg of 40-50 and 30-50 nm with a Nagg of 370-380. Even though the spherical profile can also be observed for aggregates shown in Figure 6e and f, the size distribution ranging from 60 to 80nm behaves quite different from that given in Figure 5c and is just in accordance with that of normal PEG114-b-P4VP64 micelles. As has been calculated above, the average molecular number enclosed in a water pool of E10 is 4230-4240, which is much larger than the average Nagg of normal micelles. Yang et al.30 have reported that a certain number of microspheres encapsulated in an emulsion droplet would gradually transform into a cluster during the process

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of evaporation of internal solvent. It could be inferred that all these polymer molecules assembled into more than one normal micelle in the original water pool. When the droplet was dried, micelles were forced to pack together compactly and transform into a large cluster inside a surfactant “skin”, which led to diameters ranging from 130 to 160 nm. The breakage of emulsion upon addition of acetone shucked off the thin “skin” and released all the subunits of the large cluster. Micelles were thus redispersed in the bulk solution which gave morphologies as shown in Figure 6e and f. The other method employed is named freeze/thaw-induced demulsification.40 Figure 7 shows the AFM photographs of micelles obtained by this method. For the case of E0.5, a detailed observation through TEM is presented in Figure 7b. Situations in all images come out to be consistent with those obtained by the first demulsification approach. Figure 8 provides the hydrodynamic diameter distributions of micelles obtained by freeze/thaw-induced demulsification. As calculated above, an increase in the concentrations of polymer solutions gives the confined micelles a Nagg value ranging from 40-50 to a number equal to that of a normal micelle. From Figure 8, the hydrodynamic diameters of these different confined micelles are 35.8, 44.6, and 74.1 nm. It could be clearly observed that micelles with smaller Nagg favor smaller diameters while those with bigger Nagg are more liable to show a larger size. When the number of macromolecules confined in a water cage becomes larger than the Nagg of a normal micelle, the confined micelles similar to normal ones will come into being. Besides, a gradual broadening in size distribution with the increase in Nagg could also be detected, which is in good agreement with the results provided by AFM. As we all know that most cells have sizes from a few to several thousand micrometers in diameter, in order to create a confined space of parallel size with cell, a huge emulsion up to micrometer level was also prepared. Figure 9 shows the LSCM image of the huge emulsion droplets. It can be observed that the diameters of water droplets range from 2 to 6 µm. Therefore, most of the water pools possess much more polymer chains than that required for a normal micelle. Just the same as analyzed above, it is

Li et al.

reasonable to infer that the molecules encapsulated in a water cage would probably assemble into more than one normal micelle in the huge confined space. Figure 10 presents the hydrodynamic diameter distribution of PEG114-b-P4VP64 micelles formed in huge emulsion droplets. Samples ready for examination were also obtained by freeze/ thaw-induced demulsification mentioned above. An apparent average hydrodynamic diameter of about 75.7 nm of the confined micelles also confirmed that normal micellization occurred in the micrometer sized confined spaces.

Conclusion In this study, a three-dimensional and closed microconfinement similar to an organic cell was constructed by W/O inverse emulsion as an excellent isolated domain for the self-assembly of PEG114-b-P4VP64. Through regulating the polymer concentrations of inner aqueous solutions as well as the sizes of confined spaces (emulsion droplets), the number of polymer chains encapsulated in a water pool which is equal to the aggregation number of the resulting confined micelle could be easily controlled. When Nchain< Nagg (normal micelle), confined micelles possessing different Nagg less than that of normal micelles are prepared and those with lower Nagg usually favor smaller diameters, while under the condition of Nchain > Nagg (normal micelle), normal micellization predominantly occurs and micelles similar to normal ones are preferred. Through the route we employ, as a key step toward precisely manipulating the self-assembly of block copolymers, a quasi-quantitative control over the Nagg of polymeric micelles is effectively achieved, which would definitely make pivotal progress in the development of molecular self-assembly theories and techniques, and it will be quite critical for our further exploration on precise simulation of bioaggregates in organisms. Acknowledgment. We thank the National Natural Science Foundation of China (No. 20474032, 20774051), the Program for New Century Excellent Talents in Universities, and the Outstanding Youth Fund (No. 50625310) for financial support. LA803996B