Effect of Solvent Composition on Transformation of Micelles to

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Effect of Solvent Composition on Transformation of Micelles to Vesicles of Rod-Coil Poly(n-hexyl isocyanate-block-2-vinylpyridine) Diblock Copolymers Mohammad Changez, Nam-Goo Kang, Haeng-Deog Koh, and Jae-Suk Lee* Department of Materials Science and Engineering, Department of Nanobio Materials and Electronics, Gwangju Institute of Science and Technology, 261 Cheomdan-gwagiro (Oryong-dong), Buk-gu, Gwangju 500-712, Korea Received January 9, 2010. Revised Manuscript Received March 24, 2010 The self-aggregation behavior of an amphiphilic rod-coil block copolymer of poly(n-hexyl isocyanate-block2-vinylpyridine) (PHIC189-b-P2VP228) (fP2VP = 0.78, Mn = 24.5K) in a tetrahydrofuran (THF)/water system was examined using dynamic light scattering (DLS), transmission electron microscopy (TEM), and field emission scanning electron microscopy (FE-SEM). The presence of a certain amount of water in the THF-based polymer solution induced a morphological transition from spherical solid micelles to open mouth platelike vesicles. The size of the aggregates increased with an increase in water content in the mixed solvent of THF/water. In the range of 30-40% water, the polymer formed vesicles with an interdigitated architecture of poly(n-hexyl isocyanate) (PHIC) at the center of the membrane and with the poly(2-vinylpyridine) (P2VP) block forming the outer layers and pointing toward the solvent. However, at higher water contents, the thickness of the bilayer increased due to the rearrangement of the vesicle membrane from a flip-flop to a lamellar architecture. After the degradation of the PHIC from the vesicles at basic pH, hollow spherical aggregates remained stable. After removing the THF from the mixed solvent using dialysis, large-sized compound vesicles were formed.

Introduction Unlike the conventional amphiphiles, such as low-molecularweight surfactants and lipids, block copolymers have the advantage of modifying their shape and functionality as their chemical structure, composition, size, and architecture are varied systematically.1 In selective solvents, block copolymers show a wide range of morphologies, such as spherical, ellipsoidal, hollow, or cylindrical structures, depending on both their intrinsic properties (block-block interaction parameter) and extrinsic properties (molecular weight, block composition, solvent composition, and the concentration, temperature, and pH of the solution).2 In recent years, the vesicular aggregates made of block copolymers known as polymersomes3 have attracted attention for their potential applications as cell mimicking systems,4 biosensors,5 *To whom correspondence should be addressed. E-mail: [email protected]. (1) Cornelissen, J. J. L. M.; Fischer, M.; Sommerdijk, N. A. J. M.; Nolte, R. J. M. Science 1998, 280, 1427–1430. (2) (a) Forster, S.; Antonietti, M. Adv. Mater. 1998, 10, 195–217. (b) Neiser, M. W.; Muth, S.; Kolb, U.; Harris, J. R.; Okuda, J.; Schmidt, M. Angew. Chem., Int. Ed. 2004, 43, 3192–3195. (c) Jianzhong, D.; Armes, S. P. J. Am. Chem. Soc. 2005, 127, 12800–12801. (3) Discher, B. M.; Won, Y.-Y.; Ege, D. S.; Lee, J. C.-M.; Bates, F. S.; Discher, D. E.; Hammer, D. A. Science 1999, 284, 1143–1146. (4) Koide, A.; Kishimura, A.; Osada, K.; Jang, W.-D.; Yamasaki, Y.; Kataoka, K. J. Am. Chem. Soc. 2006, 128, 5988–5989. (5) Kim, J.-M.; Ji, E.-K.; Woo, S. M.; Lee, H.; Ahn, D. J. Adv. Mater. 2003, 15, 1118–1121. (6) Vriezema, D. M.; Hoogboom, J.; Velonia, K.; Takazawa, K.; Christianen, P. C. M.; Maan, J. C.; Rowan, A. E. R.; Nolte, J. M. Angew. Chem., Int. Ed. 2003, 42, 772–776. (7) (a) Lavasanifar, A.; Samuel, J.; Kwon, G. S. Adv. Drug Delivery Rev. 2002, 54, 169–190. (b) Kabanov, A. V.; Batrakova, E. V.; Alakhov, V. Y. J. Controlled Release 2002, 82, 189–212. (c) Jeong, B.; Bae, Y. H.; Lee, D. S.; Kim, S. W. Nature 1997, 388, 860–862. (8) (a) Luo, L.; Eisenberg, A. J. Am. Chem. Soc. 2001, 123, 1012–1013. (b) Hayward, R. C.; Utada, A. S.; Dan, N.; Weitz, D. A. Langmuir 2006, 22, 4457– 4461. (c) Ding, J.; Liu, G. Macromolecules 1997, 30, 655–657. (d) Kukula, H.; Schlaad, H.; Antonietti, M.; Forster, S. J. Am. Chem. Soc. 2002, 124, 1658–1663. (e) Juan, R. H.; Lecommandoux, S. J. Am. Chem. Soc. 2005, 127, 2026–2027. (f) Kim, J. K.; Lee, J. I.; Lee, D. H. Macromol. Res. 2008, 16, 267–292.

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containers or reactors,6 and vehicles for the delivery of bioactive molecules.7 Polymersomes have been prepared from coil-coil diblock copolymers,8 peptide-based rod-coil diblocks,9 symmetric ABA-type triblock,10 and, more recently, asymmetric ABC triblock11 or grafted copolymers.12 The geometry of the supramolecular architectures of the rod-coil block copolymer is mainly governed by the volume fraction and conformational asymmetry of the rod and coil block as well as the efficiency of rod packing under the geometric constraint. An incorporation of a coil-like block into helical rod systems in a single molecular architecture may be an ideal way to create a supramolecular structure owing to its ability to segregate incompatible parts of individual molecules. Rod-coil block copolymers in a coil-selective solvent or those with a coil block much larger than the rod block have been theoretically or experimentally shown to form hockey pucks, needles, and platelike micelles rather than spherical micelles, where the rod blocks aggregate to form anisotropic cores.13,14 Block polymers with a stiff helical rodlike structure have various advantages over other synthetic block polymers because they form the secondary structures through cooperative intermolecular interactions. The rod-coil block copolymers are wellknown for their unique microphase separation; however, little is known about their associative behaviors in solution.15 (9) Hest, J. C. M. V.; Delnoye, D. A. P.; Baars, M. W. P. L.; Genderen, M. H. P. V.; Meijer, E. W. Science 1995, 268, 1592–1595. (10) Chen, X. L.; Jenekhe, S. A. Macromolecules 2000, 33, 4610–4612. (11) Liu, F.; Eisenberg, A. J. Am. Chem. Soc. 2003, 125, 15059–15064. (12) Lee, H. J.; Yang, S. R.; An, E. J.; Kim, J.-D. Macromolecules 2006, 39, 4938–4940. (13) Park, J. W.; Thomas, E. L. Macromolecules 2004, 37, 3532–3535. (14) Jenekhe, S. A.; Chen, X. L. Science 1998, 279, 1903–1907. (15) (a) Rahman, M. S.; Samal, S.; Lee, J.-S. Macromolecules 2006, 39, 5009– 5014. (b) Rahman, M. S.; Samal, S.; Lee, J.-S. Macromolecules 2007, 40, 9279–9283. (c) Rahman, M. S.; Changez, M.; Yoo, J.-W.; Lee, C. H.; Samal, S.; Lee, J.-S. Macromolecules 2008, 41, 7029–7032. (d) Koh, H.-D.; Changez, M.; Rahman, M. S.; Lee, J.-S. Langmuir 2009, 25, 7188–7192.

Published on Web 04/01/2010

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The rod-coils also form giant hollow spherical micelles or vesicles, which are much larger than the length of the rod block.16 Within these micelles or vesicles, the rods orient normal to the rod-coil interface and pack parallel to each other while the coils are stretched from the interface to minimize repulsive interactions. The formation of a giant spherical object can help to reduce the liquid crystalline deformation energy in the rod segment due to the low surface curvature of such objects. The fact that the packing behavior of the rods at the rod-coil interfaces controls the size and morphology of the resultant micelles or vesicles is of great interest. However, except for the aforementioned anisotropic micelles or hollow spheres or vesicles, little is understood regarding the solution morphology of amphiphilic rod-coil block copolymers. Recently, we reported the formation of micelles/ vesicles from a PHIC189-b-P2VP228 block copolymer in the rodselective solvent toluene. The rodlike chains (PHIC) were found to wrap tangentially to the curved interface of the spherical cores of the micelles and to the exterior of the membrane of the vesicles.17 The manipulation of the structure and composition of macromolecules at the nanometer level provides new possibilities for nanomaterial applications. In particular, micro- and nanometersized hollow particles have been the subject of intense interest due to their potential application as capsules. Recently, synthetic approaches using self-assembly strategies have been employed for the preparation of hollow aggregates. A self-assembling process is a powerful tool for the construction of a unique macromolecular architecture after degradation of the core of aggregates having a cross-linked shell.18 Herein, we report the effect of solvent composition on the morphologies of PHIC50-b-P2VP170 diblock copolymer aggregates. With increasing water contents in mixed solvent of THF/ water a spherical core-shell solid micelles transformed to bilayer polymersomes in which the PHIC rods radically align parallel to each other at the curved surface of the membrane. Furthermore, the degradation of the PHIC segment of the aggregates in the presence of a base was evaluated by UV-vis and FT-IR spectroscopy, and the morphologies of the aggregate after degradation of PHIC were evaluated using AFM and TEM without the cross-linking of the shell. From best of our knowledge, we first time report the influence of pH on the stability of nanostructure after degradation of one block segment of aggregates without stabilizing the shell by cross-linking.

Figure 1. Average hydrodynamic size of poly(n-hexyl isocyanateblock-2-vinylpyridine) (PHIC50-b-P2VP170, Mn = 24.5K, fP2VP = 0.78) block copolymer aggregates with increasing water contents in the mixed solvent of tetrahydrofuran (THF)/water at 0.5 mg/mL concentration. constant stirring at room temperature. The final polymer concentration in the solution was maintained at 0.5 mg/mL for each set of experiments. Degradation of PHIC in block copolymer aggregates was carried out in the presence of KOH. To induce block copolymer aggregation into micrometer-sized vesicles, THF from the mixed solvent of THF/water (2/8, v/v) was removed via extensive dialysis against double-distilled water. Fluorescein isothiocyanate (FITC) and rhodamine B were loaded prior to the dialysis for observation of the microvesicles by confocal laser scanning microscopy (CLSM). The hydrodynamic size of the vesicles was monitored using dynamic light scattering (DLS) at 90° at room temperature. The morphologies of the vesicles were characterized using a transmission electron microscope (TEM, JEOL 2010, Japan), operated at 120 kV, and a field emission scanning electron microscope (FE-SEM, Hitachi S-4700, Japan). For TEM observation, a 10 μL aliquot of the polymeric aggregates was deposited on a 400 mesh lacey carboncoated copper grid, dried in a desiccator, and stained using iodine vapors for 10 h. TEM image processing for inverse fast Fourier transformation (IFFT) image analysis was carried out by Digital Micrograph software (version 3.0 manufactured by Gatan Inc.).21 The polymer nanoparticles were platinum-coated on a hydrophilic silica substrate for FE-SEM analysis. Laser scanning confocal microscope images were obtained using a Radiance 2000 multiphoton confocal system (Bio-Rad) equipped with a 650 nm long-pass emission filter. Fluorescence observation was carried out at an excitation of 488 nm for FITC and 510 nm for rhodamine B.

Materials and Methods

Results and Discussion

Preparation of Aggregates. In the formation of vesicles, the preparation procedure is an important factor that largely affects the size and stability of the aggregates. Depending on the preparation condition, even the same polymeric material can form a nano- or microsized vesicle.19 The PHIC50-b-P2VP170 (Mn = 24.5K, fP2VP = 0.78) was synthesized using the living anionic polymerization process.20 The self-aggregates of the PHIC50b-P2VP170 in the mixed solvent of THF and water were prepared by first dissolving the polymer in THF, which is a common solvent for both blocks, and then adding water drop-by-drop under

Solvent-Induced Size Variation of the Aggregates. The hydrodynamic size and distribution of the aggregates as a function of the water content in the THF/water mixture are shown in Figure 1. Depending on the water content, the hydrodynamic size (Dh) of the aggregates varied from 95 ( 10 to 720 ( 30 nm. Specifically, at low water contents in THF/water mixed solvents, unimodel aggregates with ∼100 nm sizes were monitored by DLS. However, in the ranges of 10-20% water content, three different size aggregates were detected (120 ( 10, 240 ( 20, and 340 ( 20 nm). Moreover, with further increasing water content up to 80%, single aggregates with increased particle sizes of 300-800 nm were again observed. It indicated that by subsequent increasing water content in mixed solvent system induced the changes of PHIC50b-P2VP170 block copolymer morphologies from small to bigger nano-objects, and these different sizes of aggregates coexisted at 10-20% water contents.

(16) Olsen, B. D.; Segalman, R. A. Mater. Sci. Eng., R 2008, 62, 37–66. (17) Koh, H.-D.; Park, J.-W.; Rahman, M. S.; Changez, M.; Lee, J.-S. Chem. Commun. 2009, 32, 4824–4826. (18) (a) Takanobu, S, Y.; Nakatsuka, S. O.; Hideki, S. Macromolecules 2000, 33, 8524–8526. (b) Kuang, M.; Duan, H. W.; Wang, J.; Chen, D. Y.; Jiang, M. Chem. Commun. 2003, 496–497. (c) Dou, H. J.; Jiang, M.; Peng, H. S.; Chen, D. Y.; Hong, Y. Angew. Chem., Int. Ed. 2003, 42, 1516–1519. (19) Meng, F.; Hiemstra, C.; Engbers, G. H. M.; Feijen, J. Macromolecules 2003, 36, 3004–3006. (20) Shin, Y. D.; Han, S. H.; Samal, S.; Lee, J.-S. J. Polym. Sci., Polym. Chem. Ed 2005, 43, 607–615.

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(21) Kim, Y.-M.; Jeong, J.-M.; Kim, J.-G.; Kim, Y.-J.; Lim, Y. S. J. Korean Phys. Soc. 2006, 48, 250–255.

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Article Scheme 1. Mechanism for the Formation of Solid Micelles and Vesicles from Poly(n-hexyl isocyanate-block-2-vinylpyridine) Block Copolymer and Degradation of PHIC from Vesicles

Figure 2. Transmission electron microscope (TEM) micrograph of poly(n-hexyl isocyanate-block-2-vinylpyridine) (PHIC50-b-P2VP170, Mn = 24.5K, fP2VP = 0.78) block copolymer aggregates in mixed solvent of THF/water at volume ratio of (A) 95/5, (B) 80/20, and (C) 60/40. (D) Inverse fast Fourier transform (IFFT) images of the selected vesicles of (C) (marked by arrow). (E) IFFT image of the selected area of (D) which clearly showed the bilayer characteristics of the membrane. (F) Histograms shows 16.3 nm thickness of the membrane. Concentration of polymer was 0.5 mg/mL.

To investigate the correlation between the assemblies in solution and a dried sample on the substrate, the morphologies of the aggregates were examined by SEM and TEM. The morphologies of the aggregates at 5%, 20%, and 40% (v/v) water in THF are shown in Figure 2. The P2VP block, which is stained selectively with iodine vapor, appears dark in the TEM micrograph, whereas the gray zones represent the PHIC segment of the aggregates. Specifically solid micelles (30 ( 5 nm) were observed at 5% v/v water (Figure 2A), whereas the size of micelles observed by SEM was in the range of 50-80 nm (Supporting Information Figure S2A), which is much higher than the size observed from TEM. SEM provides micrographs of overall polymer structures including PHIC and P2VP domains of the micelles, while TEM image represents only the stained-P2VP domain of micelle structures. This indicates that P2VP segment of the block copolymer formed the core of solid micelles. However, with further addition of water (10% v/v) hollow aggregates along with solid micelles were clearly visible in the TEM micrograph (Supporting Information Figure 1SA). Upon further increase of the water contents (15% v/v), the block copolymer formed cylindrical aggregates and hollow structure together with solid micelles (Supporting Information Figure 1SB). In contrast, at 20% v/v water, the PHIC50-b-P2VP170 block copolymer aggregates showed characteristic bilayer vesicles with open mouths (inset Figure 2B). TEM and SEM results support the DLS observation for presence of different size aggregates in the ranges of 10-20% water contents in mixed solvent of THF/water. With increasing the water contents (20-80%), solvent became favorable for P2VP block rather than PHIC block which led to core-shell inversion and regenerate the new morphologies (vesicles) in order to compensate the increased surface free energy. Fission and fusion of the PHIC50-b-P2VP170 block copolymer vesicles were observed at 20% (v/v) or more of water, which is a common phenomenon in the presence of the common solvent for both blocks (TEM micrographs, Figure 2B,C). Fission and fusion are mainly due to an increase in the thermodynamical stability Langmuir 2010, 26(12), 9981–9985

obtained by releasing the strain from the initially formed vesicles.22 The inverse fast Fourier transform (IFFT) micrograph of a selected area of Figure 2C clearly shows the distinguishable bilayer characteristics of the vesicle membrane (Figure 2D,E). The thickness calculated from the TEM micrograph is in the range of 44 ( 3 nm. The width of the gray zone is 16.3 nm (Figure 2F) at 40% water (v/v). A PHIC-b-P2VP block copolymer has 50 units of n-hexyl isocyanate and 170 units of the 2-vinylpyridine. The length of one unit of n-hexyl isocyanate is in the range of 0.18-0.20 nm,23 and the fully extended length of a 2-vinylpyridine unit is considered to be 0.225 nm.24 Therefore, the total unit length of the PHIC rod is nearly 11 nm, and the fully extended length of the coil block (P2VP) is 38.25 nm. The average thickness of the vesicle membrane calculated from the IFFT images is 44 ( 0.3 nm (including 16.3 nm consistent with the unstained gray zone of the PHIC at the center), which is much greater than the single, stretched length of the PHIC segment of the PHIC50-b-P2VP170 block copolymer. This result suggests that the PHIC block orients radially from P2VP interface and forms an interdigitated (flip-flop) architecture at the center of the vesicle membrane, whereas the P2VP block points toward the solvent (see Scheme 1). Furthermore, the average width of the outer dark zone of the membrane is about 14 nm, which is 37% of the fully extended length of the coil. The aggregation behavior of PHIC50-b-P2VP170 diblock copolymer was observed first by dissolving it in an anhydrous THF, which is common solvent for both blocks but more favorable for PHIC and then followed by dropwise addition of water as a precipitant. The introduction of water molecules in an amphiphilic polymer solution generally led to formation of hydrogen bonds with electronegative atoms of the hydrophilic segment of the amphiphilic block copolymer, while the hydrophobic sections self-assemble by hydrophobic hydration. Association of the hydrophobic groups is opposed by the steric repulsion between the hydrated hydrophilic parts of the solute. The competitions between these two forces are mainly responsible for the formation of unique self-assembly with various shapes and sizes depending on the chemical architecture of the amphiphiles.25 THF forms an azeotrope with water which contains 6.7% water in mass fraction.26 At 5% v/v water contents in THF, PHIC50-b-P2VP170 formed only solid micelles with P2VP core due (22) Lee, J.-K.; Lentz, B. R. Biochemistry 1997, 36, 6251–6259. (23) Wu, J.; Pearce, E. M.; Kwei, T. K. Macromolecules 2002, 35, 1791–1796. (24) Lysenko, E. A.; Bronich, T. K.; Slonkina, E. V.; Eisenberg, A.; Kabanov, V. A.; Kabanov, A. V. Macromolecules 2002, 35, 6344–6350. (25) (a) Obeid, R.; Tanaka, F.; Winnik, F. M. Macromolecules 2009, 42, 5818– 5828. (b) Kujawa, P.; Tanaka, F.; Winnik, F. M. Macromolecules 2006, 39, 3048–3055. (c) Tanaka, F.; Koga, T.; Winnik, F. M. Prog. Colloid Polym. Sci. 2009, 136, 1–8. (26) Xu, S.; Wang, H. Chem. Eng. Process. 2006, 45, 954–958.

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to the presence of only one solvent system (azeotrope mixture of THF and water). However, with further addition of water in mixed solvent, the number of free water molecules tends to increase, consequently leading to change the solvent polarity. Solvent changes also tend to alter the preferred polymer architecture in the aggregates and lead to a morphological evolution from spheres to rods to vesicles as water is further on added.11,27,28 The stretching mode skeleton vibration bands for pyridine ring at 990 and 1588 cm-1 shifted to 995 and 1596 cm-1, respectively. This can be considered as an evidence for formation of hydrogen bonds29 (Supporting Information Figure S3). At 10% v/v water contents in mixed solvent, ∼3.4% v/v water is expected to be free, which is not enough to hydrate all micelles core. As a result, in a fraction of the solid micelles, the cores forming P2VP blocks can form hydrogen bond with free water which resulted in steric repulsion among the hydrated P2VP chains. Consequently, bigger hollow spherical aggregates were formed to minimize the total interfacial energy (ratio of solid/hollow micelles observed from TEM image = 6.5/1). In general, with further addition of water increases the repulsion and aggregate adapts cylindrical micelle geometry for total free energy minimization. With increasing water content, cylindrical objects can grow to significant lengths because of the interplay of the interfacial tension and the shell repulsion and then to vesicles to decrease the free energy of the system.30 However in the present case, the architecture of the vesicles clearly shows that PHIC segment of the block copolymer is present at the interior of the membrane at e20% (v/v) water contents (inset Figure 2B,E). This indicates that further addition of water in block copolymer aggregates solution induced the core (P2VP)/shell (PHIC) inversion. At high water contents (e20% (v/v)) in mixed solvent of THF/water, the hydrophobic (PHIC) block make core-shell inversion to avoid hydration and formed bilayer vesicles with PHIC interior and P2VP block pointed toward THF/water mixed solutions. Extensive network formation (background of Figure 2B,C) and vesicles aggregation were prominent at higher water contents (Figure 3) in mixed THF/ water solvent. This indicates that P2VP exterior of the vesicles membrane were highly hydrated and act as a junction point for vesicles via bridging water molecules.25 As expected, some of the vesicles collapsed in the solid state. Because THF is a good solvent for both the PHIC and P2VP blocks, at higher THF content in the mixed solvent, the P2VP block is also in direct contact with the solvent and shows a high degree of flexibility, which leads to the collapse of vesicles during THF evaporation. SEM micrographs of the aggregates at 5%, 20%, and 40% (v/v) water contents in THF are shown in the Supporting Information Figures 2SA, 2SB, and 2SC, respectively. The TEM (Figure 3A,C) and FE-SEM (Figure 3B,D) micrographs of the vesicles at higher water contents (50% and 80% v/v) show that the vesicles retained their structure in the solid state, most likely due to the glassy nature of the coil (P2VP) at low THF content in the mixed solvent. The vesicles also show an open mouth structure, indicating that they have a hollow interior, which is in good agreement with a previous report.6 The stiffness of the PHIC rod does not allow bending of the membrane or sealing of the edges as the size of the vesicles increased.6,27 The membrane thickness increased from 44 ( 0.3 to 79.5 ( 1.5 nm (27) Discher, D. E.; Eisenberg, A. Science 2002, 297, 967–973. (28) Shen, H.; Eisenberg, A. J. Phys. Chem. B 1999, 103, 9473–9487. (29) (a) Lee, J. Y.; Painter, P. C.; Coleman, M. M. Macromolecules 1988, 21, 954–960. (b) Ruokolainen, J.; Saariaho, M.; Ikkala, O.; ten Brinke, G.; Thomas, E. L. Macromolecules 1999, 32, 1152–1158. (c) Li, X.-D.; Goh, S. H. Macromol. Chem. Phys. 2003, 203, 2334–2343. (30) Shen, H.; Zhang, L.; Eisenberg, A. J. Phys. Chem. B 1997, 101, 4697–4708.

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Figure 3. (A) Transmission electron microscope (TEM) and (B) field emission scanning electron microscope (FE-SEM) micrograph of the poly(n-hexyl isocyanate-block-2-vinylpyridine) (PHIC50b-P2VP170, Mn = 24.5K, fP2VP = 0.78) block copolymer vesicle at 50% water (v/v) in mixed solvent of THF/water, respectively, (C) TEM, and (D) FE-SEM micrograph of the plate like vesicle at 80% water (v/v) in mixed solvent of THF/water. Concentration of polymer was 0.5 mg/mL.

Figure 4. (A) Degradation of PHIC from the vesicles of poly(n-hexyl isocyanate-block- 2-vinylpyridine) (PHIC50-b-P2VP170) block copolymer polymer (quantification of PHIC was carried out 252 nm) at pH = 10, (B) FT-IR spectra of PHIC50-b-P2VP170 vesicles before and after degradation, (C) transmission electron microscope micrograph of vesicles after degradation of PHIC at pH=10 (THF/water, 6/4 v/v), and (D) corresponding atomic force microscope micrograph of vesicles after PHIC degradation. Concentration of polymer was 0.5 mg/mL.

(Figure 3A,C) as the water content in the mixed solvent increased from 40% to 80% (v/v). This result suggests that the architecture of the vesicle membrane at higher water content (e50%, v/v) is rearranged and transformed from a flip-flop to a lamellar architecture as shown in Scheme 1. Degradation of PHIC in PHIC50-b-P2VP170 Block Copolymer Aggregates. The degradation of PHIC in PHIC50-bP2VP170 block copolymer aggregates was evaluated using UV-vis and FT-IR spectroscopy. Figure 4A shows the rate of degradation of PHIC in the presence of a base (pH = 10) in a THF/water (60/40% v/v) medium. FT-IR spectra of the PHIC50-b-P2VP170 Langmuir 2010, 26(12), 9981–9985

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Figure 5. (A) Field emission scanning electron microscope (FE-SEM) image of the vesicles of poly(n-hexyl isocyanate-block-2-vinylpyridine) (PHIC50-b-P2VP170, Mn = 24.5K, fP2VP = 0.78) block copolymer after removal of THF through dialysis from the mixed solvent of tetrahydrofuran/water (8/2, v/v), (B) compound vesicles, and (C) transmission electron microscope image. Confocal laser scanning micrograph of (D) the fluorescein isothiocyanate (FITC) and (E) rhodamine B loaded vesicles. CLSM micrograph of FITC loaded vesicles was obtained using 488 nm excitation and for rhodamine B loaded vesicles excitation wavelength was 510 nm. Concentration of polymer was 0.5 mg/mL.

block copolymer (with and without degradation) are shown in Figure 4B. A strong absorption band at 1700 cm-1 related to the carbonyl group of the PHIC was observed for PHIC50-b-P2VP170 and disappeared after degradation with the base. Morphologies of PHIC50-b-P2VP170 Block Copolymer Vesicles after Degradation of PHIC. Figure 4C,D represents the TEM and atomic force microscopy images of the aggregates after degradation of the PHIC from the block copolymer vesicles. The spherical nature of the aggregates remains intact and exhibits hollow micelle/vesicle characteristics in comparison to without PHIC degraded vesicles (Figure 2C, Supporting Information Figures S2C and S4A). In general, the formation of hollow micelles by the degradation of one of the segments of diblock copolymers is stabilized by the cross-linking of the shell of the aggregates.31 In the present case, due to the basic nature of P2VP, the exterior parts of the bilayer vesicle membrane, which consist of the P2VP domain of the block copolymer, remain intact under the influence of a basic medium (Scheme 1). The degraded PHIC interior segment, however, disappears, which is clearly visible in the atomic force micrograph (Figure 4D). However, at acidic pH, the vesicle morphologies were completely disintegrated, and no aggregated structure was observed either by DLS or by TEM/SEM/AFM (Supporting Information Figure S4A). At acidic pH, quaternization of the pyridine moiety is prominent, and due to charge repulsion, the P2VP segments of the vesicles are also disintegrated (Scheme 1). Compound Vesicles. THF from the THF/water mixture (20/80, v/v) was removed by extensive dialysis against the extra pure water. Fluorescent labeling of the vesicles was performed by loading with FITC and rhodamine B prior to the dialysis. The resulting vesicles showed high polydispersity, with the hydrodynamic diameter varying from 4 to 13 μm (measured by using DLS). EF-SEM and TEM images of the large vesicle are shown in parts A and B of Figure 5, respectively. The enlarged image of the (31) Chen, D. Y.; Jiang, M. Acc. Chem. Res. 2005, 38, 494–502.

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selected vesicle (marked by a box in the inset of Figure 5B) reveals the complex nature of the vesicle. The compound vesicles with diameters of 5-14 μm are shown in the CLSM images (Figure 5D,E). The hollow interior of the FITC and rhodamine B loaded compound vesicles can be easily observed in the CLSM micrograph images shown in Figure 5D,E.

Conclusion In summary, the self-aggregation behavior of an amphiphilic PHIC50-b-P2VP170 rod-coil block copolymer in a THF/water mixed solvent was examined by DLS, TEM, and FE-SEM. Depending on the water content of the solvent, the PHIC50b-P2VP170 self-assembled into configurations ranging from spherical solid micelles to bilayer vesicles. After the degradation of the PHIC from the vesicles under basic pH, the spherical nature of the aggregates remained intact and was converted to a hollow micelle/ vesicle structure. Upon removal of THF from the mixed solvent by dialysis, giant compound vesicles formed. We anticipate that the versatile nature of the pyridine group can be utilized to load various pharmaceutically active molecules for applications in biotechnology, pharmaceuticals, and cosmetics. Furthermore, the biodegradable nature of the PHIC can be used for controlled drug delivery systems. Acknowledgment. This work was partially supported by the Program for Integrated Molecular System (PIMS/GIST) and World Class University (WCU) program (Project R31-20008000-10026-0). Authors thank the Korea Basic Science Institute (KBSI), Daejon, for TEM measurements. Supporting Information Available: TEM, FE-SEM, FT-IR, and AFM of poly(n-hexyl isocyanate-block-2-vinylpyridine). This material is available free of charge via the Internet at http://pubs.acs.org.

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