Deaggregation of Water

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Reversible Thermoresponsive Aggregation/Deaggregation of Water-Dispersed Polymeric Nanospheres Exhibiting Structural Transformation Tatsuo Kaneko,†,‡,§ Kazuhiro Hamada,†,‡ Yumi Kuboshima,‡ and Mitsuru Akashi*,†,‡,§ Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita 565-0871, Department of Nanostructured and Advanced Materials, Kagoshima University, 1-21-40 Korimoto, Kagoshima 890-0065, and Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Kawaguchi Center Building, 4-1-8 Honcho, Kawaguchi 332-0012, Japan Received March 30, 2005. In Final Form: July 12, 2005 Crystalline polymeric nanospheres composed of poly{stearyl methacrylate (SMA)-co-poly(ethylene glycol) monomethacrylate (PEGm)}s were prepared by the dispersion radical polymerization of SMA and PEGm in an ethanol/water solution. Scanning electron microscopy showed that the nanospheres were highly spherical, and had a narrow size distribution. Electron spectroscopy for chemical analysis and X-ray diffraction studies of the nanospheres suggested a core-corona-type structure; the hydrophilic PEGm corona accumulated on the nanosphere surface, while the hydrophobic SMA core formed a layered structure. Heat treatment caused a melting of the SMA layers, but successive cooling allowed it to re-form. Accompanying this reversible order-disorder transition, the nanospheres also showed a reversible aggregation/deaggregation behavior in their water-dispersion state.

Introduction Polymeric particles in the nanometer size range have an extremely large specific surface area, giving them excellent material sorbability, which has attracted much attention in the fields of drug delivery and controlled release systems.1,2 Nanoparticles with a stable core of dense solid polymers such as poly(styrene) (PSt) have the special advantage of concentrating the materials absorbed by centrifugation under reasonable conditions,3 which is different from dynamic nanoparticles such as micelles,4,5 nanosized gels,6,7 and dendrimers.8-10 On the other hand, dynamic nanoparticles4,5,7,10 can absorb materials inside themselves, as well as on their surface. Nanospheres with a thermoresponsive surface have thus been developed to absorb and incrassate a large amount of materials. A nanosized hydrogel surface covered by thermoresponsive polymers with a lower critical solution temperature (LCST) such as poly(N-isopropylarylamide) (PNIPAAm) could absorb materials inside itself, and then precipitate them * To whom correspondence should be addressed. Phone: +816-6879-7356.Fax: +81-6-6879-7359.E-mail: [email protected]. † Osaka University. ‡ Kagoshima University. § JST. (1) Kataoka, K.; Harada, A.; Nagasaki, Y. Adv. Drug Delivery Rev. 2001, 47, 113. (2) Savic, R.; Luo, L.; Eisenberg, A.; Maysinger, D. Science 2003, 300, 615. (3) Akashi, M.; Niikawa, T.; Serizawa, T.; Hayakawa, T.; Baba, M. Bioconjugate Chem. 1998, 9, 50. (4) Kwon, G. S.; Okano, T. Adv. Drug Delivery Rev. 1996, 21, 107. (5) Harada, A.; Kataoka, K. Science 1999, 283, 65. (6) Pelton, R. H.; Chibante, P. Colloids Surf. 1986, 20, 247. (7) Nardin, C.; Meier, W. Chimia 2001, 55, 142. (8) Tomalia, D. A.; Baker, H.; Dewald, J.; Hall, M.; Kallos, G.; Martin, S.; Roeck, J.; Ryder, J.; Smith, P. Polym. J. 1985, 17, 117. (9) Percec, V.; Glodde, M.; Bera, T. K.; Miura, Y.; Shiyanovskaya, I.; Singer, K. D.; Balagurusamy, V. S. K.; Heiney, P. A.; Schnell, I.; Rapp, A.; Spiess, H. W.; Hudson, S. D.; Duan, H. Nature 2002, 419, 384. (10) Deng, S.; Locklin, J.; Patton, D; Baba, A; Advincula, R. C. J. Am. Chem. Soc. 2005, 127, 1744.

upon heating.11,12 This thermoresponse is reversible. Although the PNIPAAm covering method is widely used in various nanoparticles such as latex,13 polymer micelles,14,15 and inorganic particles,16-18 none of them showed thermoresponsiveness while retaining their waterdispersity characteristics, regardless of the temperature. In the present study, we prepared nanoparticles with a thermoresponsive dense core plus hydrophilic graft chains, expecting that both the thermoresponsive and water-dispersity characteristics would be retained, and collected them by centrifugation. One of the most efficient thermoresponsive characteristics is a first-order phase transition such as crystal melting, occurring very sensitively and rapidly accompanied by a large change in density.19 This first-order transition may be attractive for the nanosphere core transition. On the other hand, to our best knowledge, no nanosphere with a thermoresponsive core has ever been prepared, because the interests of most researchers have been concentrated on the surface modification of amorphous nanospheres such as polystyrene,20,21 poly(methyl methacrylate),22 and other short polymethacrylates.23,24 Here, we selected stearyl meth(11) Reese, C. E.; Mikhonin, A. V.; Kamenjicki, M.; Tikhonov, A.; Asher, S. A. J. Am. Chem. Soc. 2004, 126, 1493. (12) Kuckling, D.; Vo, C. D.; Wohlrab, S. E. Langmuir 2002, 18, 4263. (13) Chen, C. W.; Chen, M. Q.; Serizawa, T.; Akashi, M. Chem. Commun. 1998, 831. (14) Zhang, W.; Zhou, X.; Li, H.; Fang, Y.; Zhang, G. Macromolecules 2005, 38, 909. (15) Yamazaki, A.; Song, J. M.; Winnik, F. M.; Brash, J. L. Macromolecules 1998, 31, 109. (16) Deng. Y.; Yang, W.; Wang, C.; Fu, S. Adv. Mater. 2003, 15, 1729. (17) Miyazaki, A.; Nakano, Y. Langmuir 2000, 16, 7109. (18) Wang, C.; Flynn, N. T.; Langer, R. Adv. Mater. 2004, 16, 1074. (19) Mandelkern, L. J. Phys. Chem. 1971, 75, 3909. (20) Zheng, L.; Xie, A. F.; Lean, J. T. Macromolecules 2004, 37, 9954. (21) Kaneko, T.; Hamada, K.; Chen, M. Q.; Akashi, M. Macromolecules 2004, 27, 501. (22) Chen, M. Q.; Kishida, A.; Serizawa, T.; Akashi, M. J. Polym. Sci., Part A: Polym. Chem. 2000, 38, 1811. (23) Chen, M. Q.; Zhang, K.; Kaneko, T.; Liu, X.; Cai, J.; Akashi, M. Polym. J. 2005, 37, 118.

10.1021/la050836l CCC: $30.25 © 2005 American Chemical Society Published on Web 09/02/2005

Thermoresponse of Crystalline Nanospheres Scheme 1. Synthetic Scheme of Poly(SMA-co-PEGm) by Dispersion Radical Polymerization

acrylate (SMA) as a hydrophobic and thermoresponsive core component. SMA has a melting point (Tm) in the body temperature range (Tm ) 38 °C),25 and methacrylate esters have the capability of producing high molecular weight polymers.26 We prepared the poly{stearyl methacrylateco-poly(ethylene glycol) monomethacrylate} (poly(SMAco-PEGm)) by soap-free free-radical dispersion polymerization using a macromonomer; this has been termed the “macromonomer method”. The macromonomer method has produced a wide variety of nanospheres with narrow size distributions which have possible biomedical applications such as drug delivery systems,27,28 virus catchup devices,29 and vaccine carriers.30,31 Here we report the preparation of size-controlled poly(SMA-co-PEGm) nanospheres, and their thermoresponsive aggregation/ deaggregation behavior while retaining their waterdispersity characteristics based on core melting/crystallization. Experimental Section Materials. SMA (TCI), which was used as a monomer, was purified by recrystallization just before use. PEGm (numberaverage molecular weight Mn ) 4000) was a gift from Nippon Oil and Fats Co., and was used as received. 2,2′-Azobis(isobutyronitrile) (AIBN; Wako Pure Chemical Industries Ltd.), which was used as a radical initiator, was recrystallized from ethanol before use. A mixture of distilled water and ethanol (Wako) was used as the polymerization medium. Polymerization. The dispersion polymerizations of SMA and PEGm were carried out batchwise in a glass tube. The general procedure used to prepare all of the nanospheres was as follows (Scheme 1). SMA (0.962-1.000 mmol), PEGm (0.048-0.010 mmol), and AIBN (1 mol % of the total monomers) were added into the ethanol/water (13:1 v/v, 5 mL) mixture. Each polymerization batch was prepared in a glass tube, and was repeatedly degassed by freeze-thaw cycles in a vacuum apparatus, sealed off, and then placed in an incubator at 60 °C for 24 h. The resultant solutions were first dialyzed in ethanol and then in distilled water using a cellulose dialyzer tube to remove any unreacted monomer. The latex particles were then centrifuged and redispersed in water. (24) Kawaguchi, S.; Winnik, M. A.; Ito, K. Macromolecules 1995, 28, 1159. (25) Ito, K.; Usami, N.; Yayashita, Y. Macromolecules 1980, 13, 216. (26) Iwasaki, T.; Yoshida, J. Macromolecules 2005, 38, 1159. (27) Sakuma, S., Hayashi, M., Akashi, M. Adv. Drug Delivery Rev. 2001, 47, 21. (28) Sakuma, S.; Suzuki, N.; Sudo, R.; Hiwatari, K.; Kishida, A.; Akashi, M. Int. J. Pharm. 2002, 239, 185. (29) Miyake, A.; Akagi, T.; Enose, Y.; Ueno, M.; Kawamura, M.; Horiuchi, R.; Hiraishi, K.; Adachi, M.; Serizawa, T.; Narayan, O.; Akashi, M.; Baba, M.; Hayami, M. J. Med. Virol. 2004, 73, 368. (30) Akagi, T.; Kawamura, M.; Ueno, M.; Hiraishi, K.; Adachi, M.; Serizawa, T.; Akashi, M.; Baba, M. J. Med. Virol. 2003, 69, 163. (31) Kaneko, T.; Shimomai, S.; Miyazaki, M.; Baba, M.; Akashi, M. J. Biomater. Sci., Polym. Ed. 2004, 15, 661.

Langmuir, Vol. 21, No. 21, 2005 9699 Measurement. 1H NMR spectra of the terpolymers were measured using a solvent of chloroform-d with an NMR spectrometer (Varian Unity INOVA600 spectrometer) at 600 MHz. 1H NMR chemical shifts in parts per million (ppm) were recorded downfield from 0.00 ppm using tetramethylsilane (TMS) as an internal reference. Fourier transform infrared (FT-IR) spectra of the particle surface were recorded by the attenuated total reflection (ATR) method on a Perkin-Elmer Spectrum One FT-IR spectrometer after 64 scans (4 cm-1 resolution) over the range from 4600 to 400 cm-1. The molecular weight of the polymers was determined by gel permeation chromatography (GPC; Shimadzu LC-6A system with a TSK-GEL Super H2000 column), calibrated with poly(ethylene oxide) standards (eluent dimethylformamide). Electron spectroscopy for chemical analysis (ESCA) was performed with a Shimadzu ESCA 1000 apparatus employing Mg KR radiation (1253.6 eV) and a pass energy of 31.5 eV. The higher resolution utility scans were used to determine the atomic concentrations of carbon, nitrogen, and oxygen. The surface of the nanospheres was observed on a stage for scanning electron microscopy (SEM) observation with a Hitachi S-4100H SEM instrument, after gold was spattered onto the samples at a thickness of approximately 20 nm. The nanosphere size was measured by dynamic light scattering (DLS; Zetasizer Nano ZS, Malvern Instruments Ltd.) equipped with noninvasive back-scatter (NIBS) of a He-Ne laser (λ ) 632.8 nm). X-ray diffraction (XRD) patterns were taken by the Rigaku out-of-plane system for a thin film equipped with an X-ray generator (ultraX18) emitting Ni-filtered Cu KR radiation (λ ) 0.154 nm, 40 kV, 200 mA), which was monochromated by a parabolic multilayer mirror into scanning angles ranging from 2.5° to 25° at a scanning rate of 4 deg min-1. The sample was dried from the dispersion state on a washed glass plate, and was measured by reflection geometry where the X-ray incident angle was 1°. Wide-angle X-ray diffraction (WAXD) images were taken with a flat-plate camera mounted on a Rigaku ultraX18 emitting a beam the same as for XRD, and was collimated by two pinholes (L ) 0.2 mm and 0.4 mm) in transmission geometry. The nanoparticle dispersion as a sample was purged in a glass capillary (L ) 1.5 mm), and was sandwiched between two hot stages (Mettler Toledo FP82HT hot stage) with a pinhole through which the X-ray beam was transmitted. The specimen was temperature-controlled to an accuracy of (0.1° by a Mettler Toledo FP90 central processor, and was measured after being settled at a given temperature for more than 30 min.

Results and Discussion Copolymer Synthesis. The copolymerization of SMA and PEGm was performed using various molar compositions of SMA to PEGm in the feed (ratios (C) [SMA]/ [PEGm] ) 20, 39, 58, and 100) in a mixed solvent comprised of ethanol/water (13:1 v/v) at 60 °C for 24 h in vacuo as shown. SMA dissolved in ethanol well, but was insoluble in water. PEGm was soluble in both solvents. For establishing the dispersion polymerization system, we performed a preliminary experiment where water was added dropwise into an ethanol solution of SMA. We then determined the optimal solvent composition of ethanol/ water as 13:1 (v/v), which represents the maximum water composition possible for a homogeneous ethanol/water solution of SMA. The transparent monomer solution became white as a result of the polymerization reaction, to yield a colloidal solution showing a strong lightscattering phenomenon. The reaction solution was dialyzed for 3 days in ethanol and pure water to remove any unreacted monomers. The products were then gathered by centrifugation, and the molecular structure of the powder was analyzed spectroscopically to confirm the generation of poly(SMA-co-PEGm)s. Figure 1a shows a sample FT-IR/ATR spectrum of a polymer with a C of 58,

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Figure 1. FT-IR (a) and 1H NMR (b) spectra of poly(SMA-co-PEGm) with an SMA/PEGm composition in the feed of 58. Table 1. Particle Formation by Copolymerization of Stearyl Methacrylate with PEG Macromonomera composition no.

SMA amt (mmol)

PEGm amt (mmol)

[SMA]/[PEGm] in feed

SMA:PEGm in copolymb

1 2 3 4

0.962 0.985 0.993 1.000

0.048 0.025 0.017 0.010

20 39 58 100

98.6:1.4 99.3:0.7 99.5:0.5 99.6:0.4

molecular weightsc Mn × 104 Mw × 105 Mn/Mw 2.5 4.4 4.0 4.3

1.5 1.5 1.4 1.5

6.2 3.4 3.5 3.4

yield (%) 38 49 33 40

particl e size (CV)d [nm (%)] at 20 °C at 60 °C at 30 °C 540 (27) 835 (26) 1480 (49) 2300 (53)

530 (16) 830 (14) 955 (14) 1360 (11)

535 (26) 835 (22) 1300 (39) 1480 (50)

a SMA and PEGm refer to stearyl methacrylate and poly(ethylene glycol) monomethoxy methacrylate, respectively. The reaction period was 24 h. The temperature was 60 °C. The solvent was a mixture of water and ethanol (water:ethanol ) 1:13 v/v). The Mn of PEGm is 4000. b Determined by 1H NMR spectra. c Measured by GPC in a PEG standard. d Measured by dynamic light scattering at various temperatures.

which represents the vibration of the alkyl groups (1463 and 2917 cm-1), ether groups (1062 cm-1), and ester groups (1728 and 1146 cm-1), confirming that these samples were both composed of monomeric units. The 1H NMR spectra also confirmed the copolymer formation seen in the representative spectrum of the polymer with a C of 58 (Figure 1b); there was a PEG methylene proton peak at a chemical shift of 3.64 ppm, long alkyl proton peaks at 0.88, 1.26, 1.55, and 3.91 ppm, and main chain proton peaks in the range of 1.02-2.21 ppm. Furthermore, we confirmed the complete removal of unreacted monomers from the products by the absence of methacrylate proton peaks around 5.6 or 6.2 ppm (Figure 1b). Using the 1H NMR spectra, we could estimate the molar ratios of the monomer units in the copolymers from the integration ratios of the PEGm methylene peak to the total aliphatic peaks from SMA and the main chain. The FT-IR and NMR spectra of the other copolymers showed diagrams similar to those in Figure 1, and the calculations of the copolymer composition are summarized in Table 1. One can see that the ratios of PEGm to SMA were in the range of 1.4-0.4, which was much lower than in the feed. These results suggest that PEGm was less reactive than SMA, presumably due to the bulky oligomeric chains. The copolymers dissolved in dichloromethane, tetrahydrofuran, and hex-

ane, but could be dispersed in water, ethanol, acetone, dimethyl sulfoxide, and dimethylformamide, regardless of the C ratio. The yields of the copolymers were 33-49 wt % with respect to both monomers. The number-average molecular weight, Mn, and the weight-average molecular weight, Mw, of the copolymers were 2.5 × 104 to 4.4 × 104 and 1.4 × 105 to 1.5 × 105, respectively. The resulting nanospheres showed a wide molecular weight distribution ratio (Mw/Mn) in the range of 3.4-6.2. Similar results have been reported in previous papers on the different copolymers composed of hydrophobic monomer and hydrophilic macromonomer units.21 To clarify the effects of solution nonhomogeneity on the copolymer composition, we also made a homogeneous copolymerization of SMA (0.993 mol) and PEG macromonomer (0.017 mmol) in tetrahydrofuran, and the results were compared with those of the no. 3 system in Table 1. The reaction solution was put into a large amount of ethanol to precipitate the copolymer product (yield 60%). An 1H NMR study of the copolymer showed that the SMA unit composition to the total units was 1.0 mol %, which is higher than the ratio of copolymer nanospheres prepared in the ethanol/water nonhomogeneous system. The copolymer prepared in the THF homogeneous solution did not transform into nanospheres in ethanol or water despite the enhanced incorporation of

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Figure 2. SEM images of poly(SMA-co-PEGm) nanospheres with SMA/PEGm compositions in the feed of 20 (a), 39 (b), 58 (c), and 100 (d), and representative ESCA diagrams of the nanospheres (SEM, a) (e).

the solvatophilic PEG macromonomer units into the copolymer chains. This result showed the importance of the solution nonhomogeneity for preparing the waterdispersed nanospheres, presumably forming the structure of an SMA-rich core plus a PEG-rich corona. The Mn, Mw, and Mn/Mw (GPC) were 8.2 × 104, 5.2 × 105, and 6.5, respectively. The molecular weights, polydispersity, and yield of the copolymer in the homogeneous system were higher than those in the nonhomogeneous system (no. 3 in Table 1). This may be due to the increased composition of PEG macromonomer units with a much higher molecular weight than the SMA units. The reason for the low composition of PEG macromonomer in the nanospheres may be the preferential incorporation of SMA monomers into the nanospheres due to the solvatophobic effects. Preparation and Structure of Nanospheres. Waterdispersed copolymers with various C ratios were dried in vacuo, and were observed by SEM. Figure 2a-d shows the formation of spherical nanoparticles from the copolymers. The nanosphere diameters were estimated at 320390 nm for a C of 20, 500-640 nm for a C of 39, 610-710 nm for a C of 58, and 570-710 nm for a C of 100. With increasing C values, the size distribution seemed to become narrower. We speculated that SMA may react preferentially during the early stages of the polymerization to give SMA-rich chains with a larger Mn, whereas PEGm may react subsequently to give PEGm-rich chains with a smaller Mn. Figure 2e shows a representative ESCA diagram of a poly(SMA-co-PEGm) nanosphere with a C of 20, indicating a distinct O 1s peak at around 535.0 eV as well as a C 1s peak at around 286.5 eV, corresponding to the C-O binding energy, whereas the C 1s peak at around 285 eV corresponding to the C-C binding energy was very small. This result suggests that the surface accumulation of PEGm chains on the nanospheres buried the alkyl backbones and stearyl side chains inside. Our previous studies have demonstrated that the dispersion radical polymerization of hydrophobic monomers such as styrene (St)32 and methyl methacrylate (MMA)22 in the presence of PEGm in an ethanol/water solvent formed a core-corona nanosphere structure, i.e., a hydrophobic core and the PEGm corona chain grafting on the surface, regardless of the size or molecular composition. In the present study, we first prepared core-corona nanospheres (32) Serizawa, T.; Takehara, S.; Akashi, M. Macromolecules 2000, 33, 1759.

Figure 3. Size distribution of poly(SMA-co-PEGm) nanospheres with SMA/PEGm compositions in the feed of 20 (a), 39 (b), 58 (c), and 100 (d). The symbols O, 0, and 4 refer to measured temperatures of 20, 60, and 30 °C after cooling from 60 °C, respectively.

with a hydrophilic corona of PEG and a hydrophobic core of long alkyl side chains. The size of the nanospheres in the water-dispersed state was measured by DLS, and Figure 3 shows the size distribution diagrams. The maximum peak diameter (dm) and the coefficients of variation (CV; %) are summarized in Table 1. In addition, the dm was plotted against the C value in Figure 4. The dm values of the nanospheres at 20 °C (open circles) increased with increasing C values in correspondence with the SEM results, and were higher than the diameters estimated in the SEM study. With respect to the size distribution, the CV values were 2653%, and were unexpectedly high in contrast with the SEM results. On the basis of these results, we can speculate that some parts of the nanospheres may have aggregated to give a wide size distribution and a high dm in the dispersion state.

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Figure 4. Changes in the particle size of poly(SMA-co-PEGm) nanospheres as a function of the SMA/PEGm composition in the feed, measured at various temperatures.

Figure 5. X-ray diffraction diagram of poly(SMA-co-PEGm) nanospheres with an SMA/PEGm composition in the feed of 20. L1, L2, and L3 represent diffractions of a layered structure. The black arrow shows a peak from the main diffraction for the SMA crystal, while the gray arrows show the shoulders of the main diffractions from the PEGm crystal.

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Figure 6. DSC thermogram of poly(SMA-co-PEGm) nanospheres with an SMA/PEGm composition in the feed of 20. The black arrows show a peak from a melting endotherm for the SMA crystal, while a gray arrow shows the melting endotherm for the PEGm crystal.

Figure 7. Wide-angle X-ray diffraction image of poly(SMAco-PEGm) nanospheres with an SMA/PEGm composition in the feed of 20, taken in the water-dispersed state at various temperatures. The black arrows show a main diffraction arc for the SMA crystal, while the gray arrows show main diffraction arcs for the PEGm crystal.

Since long alkyl chains composed of more than 14 carbons can show a strong stacking behavior to form a crystalline structure at ambient temperature,33 we investigated the structure of the poly(SMA-co-PEGm) nanospheres. Figure 5 shows a representative XRD diagram of nanospheres with a C of 20. Several distinct diffractions appeared at around 2θ ) 2.9° (L1, shoulder), 5.5-6.2° (L2), 8.6-9.6° (L3), and 20.5-22.1° (dSMA) (θ ) diffraction angle), corresponding to spacings of d ) 3.0, 1.4-1.6, 0.9-1.0, and 0.40-0.43 nm, respectively (Figure 3c). Three diffractions located in the smaller angle range (L1, L2, L3) showed a clear relationship of their spacings as 1:2:3, which indicates the formation of a layered structure with a thickness of dL ) 2.7-3.2 nm. Assuming that the SMA side chains adopted a fully extended alltrans conformation aligning perpendicularly to the main chains, we calculated the sum of the main chain thickness plus the side chain length of poly(SMA-co-PEGm)s at 2.6 nm, which is close to the dL thickness, although it is slightly shorter. The dSMA corresponds to a side-by-side arrangement of the stearyl groups according to the literature.34 These results indicate that the stearyl side chains formed a layered structure in which the side chains stacked regularly side-by-side. Since SMA is very hydrophobic, this layered structure should be formed inside the nanosphere. In addition, two shoulders around the dSMA peak appeared at 2θ ) 19.3° and 23.6°, corresponding to spacings of 0.46 and 0.38 nm. These shoulders can be

assigned to PEGm crystals, since PEG shows crystalline peaks at diffraction angles of 2θ ) 19.7° and 23.9°.35 Thermoresponsive Behavior. We macroscopically observed a turbidity change in the nanosphere dispersion solution upon heating, but the nanospheres retained their colloidal dispersion stability regardless of the temperature. We then investigated the thermoresponsive properties of the nanospheres in the dry and water-dispersion states. Figure 6 shows representative DSC curves of the nanospheres with a C of 20 in the dry and water-dispersed states. Nanospheres in the dried power state showed two endotherms at 39 and 54 °C which correspond to the melting of SMA and PEG crystals, respectively, according to the literature.26,36 On the other hand, the endotherm at 54 °C disappeared in the water-dispersion state. Other samples showed the same phenomenon. This may be due to the breaking of the PEG crystals by hydration in water. We then performed an additional XRD study of the nanospheres in the water-dispersion state at various temperatures. Figure 7 shows WAXD images of waterdispersed nanospheres with a C of 20. One can see that there are three diffraction arcs in the WAXD image taken at 29 °C. The black arrow shows the diffraction of the SMA crystal, whereas the gray arrows show the two diffractions of the PEGm crystals in Figure 7a. This image indicates that neither crystal was broken by the waterdispersion treatment. When the temperature increased to 40 °C, the SMA diffraction disappeared while the PEGm crystalline diffraction remained (Figure 7b), indicating that the SMA crystals showed melting behavior in the water-dispersion state. At 60 °C, no crystalline diffraction

(33) Miyazaki, T.; Kaneko, T.; Gong, J. P.; Osada, Y. Macromoelcules 2001, 34, 6024. (34) Matsuda, A.; Sato, J.; Yasunaga, H.; Osada, Y. Macromolecules 1994, 27, 7695.

(35) Tadokoro, H.; Chatani, Y.; Yoshimura, T.; Tahara, S.; Murahashi, S. Makromol. Chem. 1964, 74, 109. (36) Tanaka, S.; Ogura, A.; Kaneko, T.; Murata, Y.; Akashi, M. Macromolecules 2004, 37, 1370.

Thermoresponse of Crystalline Nanospheres

Figure 8. Schematic illustration of the thermoresponsive deaggregation/reaggregation behavior accompanying the transformation of the inner structures for poly(SMA-co-PEGm) nanospheres.

appeared but a broad halo appeared (Figure 7c), indicating that the PEGm crystals disappeared to give rise to liquid nanospheres. The DSC thermogram showed no PEGm crystalline melting in the water-dispersion state (Figure 6), which suggests that the PEGm crystals did not melt upon heating but dissolved in water by coming out of the molten core. When the nanospheres were cooled to 30 °C from 60 °C (Figure 1d), only the SMA diffraction reappeared, indicating that the SMA crystals re-formed but the PEGm crystals did not. The WAXD study demonstrated that the poly(SMA-co-PEGm) nanospheres showed a reversible order-disorder transition. The effects of the order-disorder transition on the water dispersibility of the nanospheres were investigated. As seen in Figures 3 and 4, the dm of the nanospheres changed upon a temperature change. At C ) 58 and 100, the dm at 60 °C decreased remarkably, but increased again at 30 °C after cooling. Moreover, as a result of heating from 20 to 60 °C, the CV values of all nanospheres prepared here became very low at 11-16%, which represents a very narrow distribution of particle size similar to that of the previously reported nanospheres prepared by the analogous dispersion radical polymerization of St or MMA with the PEGm macromonomer. This result strongly suggests that the aggregated nanospheres in water were disaggregated upon heating to give rise to a narrow size distribution, which was comparable with the SEM results. However, successive cooling to 30 °C allowed the nanospheres to reaggregate, although the CV values were slightly smaller than the initial values. This thermoresponsive aggregation/deaggregation behavior may be associated with the reversible orderdisorder transition of the nanospheres. A tentative illustration of this thermoresponsive structural transformation is shown in Figure 8. If the polymerization process were based on the mechanism of conventional dispersion polymerization,37 then the SMA-rich polymers would form the nanosphere core at the beginning of the polymerization, and most of the PEGm macromonomers would subsequently cover the core, since they have high solvent affinity in water/ethanol. The surface layer may then stabilize the nanosphere dispersion state, as previously reported in the poly(St-co-PEGm) system, where the St-rich chains formed the hydrophobic core and the PEGm chains covered the core as a grafted corona.32 In the poly(SMA-co-PEGm) nanosphere, WAXD studies (37) Fitch, R. M.; Tsai, C. H. J. Polym. Sci., Polym. Lett. Ed. 1970, 703, 8.

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demonstrated the crystallization of both SMA and PEGm units in the water-dispersion state at 20 °C despite the hydrophilic nature of PEGm. We can hypothesize on the basis of the poly(St-co-PEGm) structure that some of the PEGm chains are crystallized around the surface inside the SMA-rich core, while the other chains are hydrated as a corona chain grafting onto the nanosphere surface. The SMA units formed the layered structure whose spacing in some cases was higher than the full length of SMA, presumably due to the presence of long PEGm chains which may have crystallized between the SMA layers as shown at the bottom of Figure 8a. Crystalline nanospheres composed of poly(SMA-co-PEGm)s are easier to aggregate than amorphous ones composed of poly(MMA-co-PEGm)s and poly(St-co-PEGm)s. The reason for this phenomenon is difficult to understand, but one possible explanation is that the aggregation is associated with the nanosphere surface force, which may be strong in crystallizable molecules. When the crystals inside the core are melted by heating to 60 °C, an oil-in-water colloidal system may evolve where the PEG chains function at the colloidal interface as stabilizers (Figure 8b). If some of the hydrophilic PEGm chains come out of the surface from the SMA domains in the molten state, then the surface density of the PEGm chains may increase. As a result, the surface hydrophilicity of the nanospheres increases to make them disaggregate. The successful preparation of monodistributed nanospheres at 60 °C may be attributed to dispersion polymerization in the disaggregated state. The dm measured in the water-dispersion state by DLS at 60 °C is a bit higher than the size measured by SEM observation of the dried sample, presumably due to the water-swelling of the PEGm chain and the volume change induced by the core melting. Following successive cooling, the SMA units were recrystallized; the surface force might recover, but the PEGm number on the surface might not. The dm and CV values also did not recover completely. Although the PEGm units remaining in the core may be recrystallized, this amount was too small to detect by WAXD imaging. Thus, the order-disorder transition based on the melting of the SMA layered structure induced the aggregation/ deaggregation phenomenon of the nanospheres. Conclusions Crystalline polymeric nanospheres of poly(SMA-coPEGm)s were prepared by dispersion radical polymerization in an ethanol/water solution. ESCA and XRD studies of the nanospheres suggested a core-corona structure; a hydrophilic PEGm corona encasing a hydrophobic SMA core formed the layered structure. Temperature changes resulted in a reversible order-disorder transition based on the melting of the SMA layers. Accompanying this transition, the nanospheres showed an aggregation/deaggregation behavior in the waterdispersion state. We believe that this transition is very efficient because crystal melting is a first-order transition occurring via the cooperation of whole molecules. The oil domains water-dispersing stably above the melting temperature of the core may absorb the low-polarity molecules, and thus hold them following crystallization. Therefore, the molecule-incorporated nanosphere may function efficiently as a thermoresponsive molecule-releasing system. Acknowledgment. We show deep appreciation to Dr. Toshiko Muneishi (Osaka University) for measuring the NMR spectra and for valuable discussions. LA050836L