Thermoresponsive Vesicular Morphologies Obtained by Self

pubs.acs.org/Langmuir © 2009 American Chemical Society

Thermoresponsive Vesicular Morphologies Obtained by Self-Assemblies of Hybrid Oligosaccharide-block-poly(N-isopropylacrylamide) Copolymer Systems Issei Otsuka,† Keita Fuchise,‡ Sami Halila,† S
ebastien Fort,† Karim Aissou,† Isabelle Pignot-Paintrand,† Yougen Chen,‡ Atsushi Narumi,§ Toyoji Kakuchi,‡ and Redouane Borsali*,† † Centre de Recherche sur les Macromol
ecules V
eg
etales (CERMAV, UPR-CNRS 5301) affiliated with Universit
e Joseph Fourier and member of Institut de Chimie Mol
eculaire ICMG FR2607, BP53, 38041 Grenoble Cedex 9, France, ‡Division of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University, 060-8628 Sapporo, Japan, and §Department of Polymer Science and Engineering, Graduate school of Science and Engineering, Yamagata University, 992-8510 Yonezawa, Japan

Received July 26, 2009. Revised Manuscript Received September 11, 2009 This work discusses the self-assembly properties of thermoresponsive hybrid oligosaccharide-block-poly(N-isopropylacrylamide) copolymer systems: maltoheptaose-block-poly(N-isopropylacrylamide) (Mal7-b-PNIPAMn) copolymers. Those systems at different molar masses and volume fractions were synthesized using Cu(I)-catalyzed 1, 3-dipolar azide/alkyne cycloaddition, so-called “click” chemistry, between an alkynyl-functionalized maltoheptaose (1) and poly(N-isopropylacrylamide) having a terminal azido group (N3-PNIPAMn) prepared by atom transfer radical polymerization (ATRP). While the cloud point (Tcp) of the N3-PNIPAMn ranged from 36.4 to 51.5 °C depending on the degree of polymerization, those obtained of the diblock copolymers ranged from 39.4 to 73.9 °C. The self-assembly of such systems is favored due to the hydrophobicity of the PNIPAM in water above the Tcp. While the N3-PNIPAMn present polydisperse globular shape with a mean diameter of 500 nm, well-defined vesicular morphologies with an approximate diameter of 300 nm are obtained in diblock copolymer systems. These results were obtained and confirmed using static and dynamic light scattering as well as imaging techniques such as transmission electron microscope experiments.

Introduction The ability of amphiphilic block copolymers to self-assemble into nano-organized morphologies (micelles, vesicles, bilayers, etc.) in aqueous and organic media has been widely studied.1-4 In recent years, the interest in the self-assembly at the nanoscale of “hybrid” block copolymers, based on a biodegradable natural block and a synthetic polymer block, has been growing for their potential applications in biomedical use, such as gene and drug deliveries.5,6 Carbohydrates are one of the abundant raw materials for the natural block that show biodegradability, biocompatibility, and biorecognition ability. Although this topic is of great importance, there have been few reports on the synthesis of saccharide-based hybrid block copolymers involving different strategies. In the early 1980s, a pioneering study in this field was carried out by Ziegast and Pfannem€uller on the synthesis of poly(ethylene oxide)-block-oligosaccharide structures by end-toend coupling techniques.7 Since then, several studies on the synthesis of block copolymers consisting of polysaccharides, such *Corresponding author: E-mail [email protected]; Fax +33 476 037 640. (1) Darling, S. B. Prog. Polym. Sci. 2007, 32, 1152–1204. (2) Rodrı´ guez-Hern
andez, J.; Ch
ecot, F.; Gnanou, Y.; Lecommandoux, S. Prog. Polym. Sci. 2005, 30, 691–724. (3) Riess, G. Prog. Polym. Sci. 2003, 28, 1107–1170. (4) Hamley, I. W. Nanotechnology 2003, 14, R39–R54. (5) Blanazs, A.; Armes, S. P.; Ryan, A. J. Macromol. Rapid Commun. 2009, 30, 267–277. (6) Mecke, A.; Dittrich, C.; Meier, W. Soft Matter 2006, 2, 751–759. (7) Ziegast, G.; Pfannem€uller, B. Makromol. Chem., Rapid Commun. 1984, 5, 373–379.
(8) Bosker, W. T. E.; Agoston, K.; Cohen Stuart, M. A.; Norde, W.; Timmermans, J. W.; Slaghek, T. M. Macromolecules 2003, 36, 1982–1987.

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as dextran,8-13 hyaluronan,14,15 and amylose,16-19 with several synthetic blocks have been reported, using enzymatic polymerization, coupling techniques, and living radical polymerization. In recent years, there has been increasing interest in “click” chemistry to design block copolymers.20 For instance, Lecommandoux and co-workers reported a simple and versatile strategy for synthesizing block copolymers consisting of polysaccharides and polypeptides by using Cu(I)-catalyzed 1,3-dipolar azide/ alkyne cycloaddition.13,15 This strategy has many advantages compared to the others, and more precisely, it circumvents the incompatibility problem between saccharides and other blocks in terms of low coupling reactivity. Because of the amphiphilic nature of these polysaccharide-based hybrid block copolymers, recent work highlights the existence of expected self-assembled nanoparticles: micellar or vesicular morphologies obtained both (9) Liu, J.-Y.; Zhang, L.-M. Carbohydr. Polym. 2007, 69, 196–201. (10) Hernandez, O. S.; Soliman, G. M.; Winnik, F. M. Polymer 2007, 48, 921– 930. (11) Houga, C.; Le Meins, J.-F.; Borsali, R.; Taton, D.; Gnanou, Y. Chem. Commun. 2007, 3063–3065. (12) Houga, C.; Giermanska, J.; Lecommandoux, S.; Borsali, R.; Taton, D.; Gnanou, Y.; Le Meins, J.-F. Biomacromolecules 2009, 10, 32–40. (13) Schatz, C.; Louguet, S.; Le Meins, J.-F.; Lecommandoux, S. Angew. Chem., Int. Ed. 2009, 48, 2572–2575. (14) Yang, Y.; Kataoka, K.; Winnik, F. M. Macromolecules 2005, 38, 2043– 2046. (15) Upadhyay, K. K.; Le Meins, J.-F.; Misra, A.; Voisin, P.; Bouchaud, V.; Ibarboure, E.; Schatz, C.; Lecommandoux, S. Biomacromolecules, in press. (16) Akiyoshi, K.; Kohara, M.; Ito, K.; Kitamura, S.; Sunamoto, J. Macromol. Rapid Commun. 1999, 20, 112–115. (17) Loos, K.; Stadler, R. Macromolecules 1997, 30, 7641–7643. (18) Loos, K.; M€uller, A. H. E. Biomacromolecules 2002, 3, 368–373. (19) Loos, K.; B€oker, A.; Zettl, H.; Zhang, M.; Krausch, G.; M€uller, A. H. E. Macromolecules 2005, 38, 873–879. (20) Lutz, J.-F. Angew. Chem., Int. Ed. 2007, 46, 1018–1025.

Published on Web 10/15/2009

DOI: 10.1021/la902743y

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in organic and in aqueous media.9-13,15,19 However, very few attempts have been made to control these morphologies by means of external stimuli, which should be of great importance and significant for a controlled molecular encapsulation/release system.10,15 Hence, constructing a new hybrid block copolymer containing saccharidic and stimuli-responsive polymer blocks represents a step forward toward new class of amphiphilic natural-based block copolymer systems. Herein, we demonstrate (1) precise synthesis of hybrid block copolymers containing poly(N-isopropylacrylamide) (PNIPAM) and maltoheptaose blocks by combining ATRP and “click” chemistry and (2) study of their thermoresponsive self-assemblies in water. PNIPAM is a representative thermoresponsive polymer that shows sharp phase transition in water due to its coil/globule transition involving conformational change at a temperature defined as the lower critical solution temperature (LCST).21,22 Thus, the obtained diblock copolymers will be amphiphilic above the LCST of PNIPAM block, and their self-assembled structures should be controlled by changing temperature.23-25 Maltoheptaose is a renewable oligosaccharide derived from starch, which is composed of seven R-1,4-linked glucopyranosyl units. In sharp contrast to the other polysaccharides derived from starch, maltoheptaose has monodisperse molecular weight. Consequently, this feature will lead to the synthesis of a relatively narrow molar mass distribution of hybrid block copolymers, which should afford well-organized morphologies. To the best of our knowledge, this is the first study on the synthesis of well-defined oligosaccharide-block-PNIPAM copolymers by a “convenient” approach using “click” chemistry and their thermoresponsive self-assembly properties.

Experimental Section Materials. Maltoheptaose was kindly supplied from Hayashibara Biochemical Laboratories, Inc., Japan, and used as received. N-Isopropylacrylamide (NIPAM) was kindly supplied from KOHJIN Co., Japan, recrystallized from hexane/toluene (10:1, v/v), and stored in an inert atmosphere at -30 °C. Tris[2-(dimethylamino)ethyl]amine (Me6TREN) was kindly supplied from Mitsubishi Chemical Co., Japan, and distilled over CaH2 prior to use. Propargylamine, L-(+)-ascorbic acid, and copper(II) sulfate pentahydrate were purchased from Fluka Chemical Corp. with high purity (g98%) and used as received. Isopropyl alcohol (HPLC grade, g99.7%) was purchased from Kanto Chemical Co., Japan, and used as received. Methanol and acetic anhydride were purchased from Solvent Documentation Synthese (SDS), France, with analytical grade quality and used as received. N-(20 -Azidoethyl)-2-chloropropionamide (AECP) was synthesized according to the literature.26 Synthesis of N-Maltoheptaosyl-3-acetamido-1-propyne (1). A suspension of maltoheptaose (10.0 g, 8.67 mmol) in neat propargylamine (11.9 mL, 174 mmol) was stirred vigorously at room temperature until complete conversion of the starting material (72 h), checked by TLC (eluent: BuOH/EtOH/H2O = 1/3/1). After complete disappearance of the starting material, the reacting mixture was dissolved in methanol (100 mL) and then precipitated in CH2Cl2 (300 mL). The solid was filtrated and washed with a mixture of MeOH and CH2Cl2 (MeOH:CH2Cl2 = (21) Scarpa, J. S.; Mueller, D. D.; Klotz, I. M. J. Am. Chem. Soc. 1967, 89, 6024– 6030. (22) Heskins, M.; Guillet, J. E. J. Macromol. Sci., Chem. A 1968, 2, 1441. (23) Qin, S.; Geng, Y.; Discher, D. E.; Yang, S. Adv. Mater. 2006, 18, 2905–2909. (24) Li, Y.; Lokitz, B. S.; McCormick, C. L. Angew. Chem., Int. Ed. 2006, 45, 5792–5795. (25) Chen, X.; Ding, X.; Zheng, Z.; Peng, Y. New J. Chem. 2006, 30, 577–582. (26) Narumi, A.; Fuchise, K.; Kakuchi, R.; Toda, A.; Satoh, T.; Kawaguchi, S.; Sugiyama, K.; Hirao, A.; Kakuchi, T. Macromol. Rapid Commun. 2008, 29, 1126– 1133.

2326 DOI: 10.1021/la902743y

1:3, v/v, 300 mL). A solution of acetic anhydride in MeOH (acetic anhydride:MeOH = 1:20, v/v, 1 L) was added to the solid and stirred overnight at room temperature. After complete disappearance of the starting material checked by TLC (eluent: CH3CN/ H2O = 13/7), the solvent of the mixture was evaporated, and the traces of acetic anhydride were removed by coevaporation with a mixture of toluene and methanol (1:1, v/v). The resulting solid was dissolved in water and lyophilized to afford 1 as a white solid (8.75 g, 78%). Rf = 0.34 (13:7, CH3CN-H2O). 1H NMR (D2O): δ 5.46 and 5.00 (2  d, 1H, rotamers, J1-2 = 9.20 Hz and J1-2 = 8.87 Hz, H-1GlcI), 5.36-5.31 (m, 6H, H-1GlcII-GlcVII), 4.24-3.30 (m, 44H, H-2, 3, 4, 5, 6a, 6bGlcI-GlcVII, and NCH2), 2.66 and 2.50 (2  s, 1H, rotamers, CtCH), 2.24 and 2.16 (2  s, 3H, rotamers, NCOCH3). 13C NMR (D2O): δ 176.22, 175.04, 100.0999.76, 86.80, 82.03, 80.26, 79.64, 77.47, 77.20-76.85, 76.38, 76.23, 73.68, 73.23, 73.08, 72.10, 72.06, 71.90, 71.85, 71.54, 70.58, 70.08, 69.69, 60.84, 60.78, 33.19, 30.44, 21.98, 21.51. HRMS ESI-TOF (m/z) Calcd for [M + Na]+: 1254.4123. Found: 1254.4122.

Synthesis of PNIPAM with an Azide End-Group (N3PNIPAMn). A typical polymerization method is as follows: to

NIPAM (50.0 g, 442 mmol) and CuCl (0.220 g, 2.21 mmol) in a three-neck flask was added degassed isopropyl alcohol (55 mL) under an argon atmosphere. After sealing, a degassed Me6TREN solution in isopropyl alcohol (2.95 M, 0.75 mL) was added to the flask and stirred at 20 °C for 10 min. To the mixture was added a degassed AECP solution in isopropyl alcohol (2.95 M, 0.75 mL) and stirred at 20 °C for 4 h. The polymerization was stopped by exposure to air. The NIPAM conversion was directly determined from the 1H NMR spectrum of the aliquots of the mixture in CDCl3 (93%). The reaction mixture was diluted with tetrahydrofuran (THF) and passed through an alumina column to remove the copper complex. The eluent was dialyzed using a cellophane tube (Spectra/Por 6 Membrane; MWCO: 1000) in methanol and then evaporated. The residue was dissolved in a small amount of THF and then precipitated in a large amount of hexane at 0 °C. The precipitate was filtrated and dried in vacuo to afford N3-PNIPAM220 as a white solid (50.4 g). Mn,SEC = 34 900, Mw/Mn = 1.25.

Synthesis of Maltoheptaose-block-poly(N-isopropylacrylamide) (Mal7-b-PNIPAMn). To a solution of N3-PNIPAM220

(2.00 g, 5.73  10-5 mol, 1 equiv), 1 (635 mg, 5.15  10-4 mol, 9 equiv), and sodium ascorbate (126 mg, 6.36  10-4 mol, 11 equiv) in water (30 mL) was added a 5.73  10-2 M solution of CuSO4 (1 mL, 5.73  10-5 mol, 1 equiv). The mixture was stirred at room temperature for 48 h until the IR spectra showed complete disappearance of the signal due to the azide group of starting material. The mixture was first dialyzed against water using a cellophane tube (Spectra/Por 6 Membrane; MWCO: 2000) and then freeze-dried to afford Mal7-b-PNIPAM220 as a white solid (2.04 g). Experimental Equipment. The 1H NMR and 13C NMR spectra were recorded using a 400 MHz Bruker Avance DRX400. The size exclusion chromatography (SEC) of N3-PNIPAMn was performed at 40 °C using a Jasco highperformance liquid chromatography (HPLC) system (PU-980 Intelligent HPLC pump, CO-965 Column oven, RI-930 Intelligent RI detector, and Shodex DEGAS KT-16) equipped with a Shodex Asahipak GF-310 HQ column (linear, 7.6 mm  300 mm; pore size, 20 nm; bead size, 5 μm; exclusion limit, 4  104) and a Shodex Asahipak GF-7 M HQ column (linear, 7.6 mm  300 mm; pore size, 20 nm; bead size, 9 μm; exclusion limit, 4  107) in DMF containing lithium chloride (0.01 M) at the flow rate of 0.4 mL min-1. The number-average molecular weight (Mn) and polydispersity index (Mw/Mn) of N3-PNIPAMn were determined on the basis of a polystyrene calibration. The SEC of N3-PNIPAMn and Mal7-b-PNIPAMn were performed at 30 °C using a Waters Alliance GPCV2000 system equipped with three detectors online: a differential refractometer, a viscometric detector, Langmuir 2010, 26(4), 2325–2332

Otsuka et al. and a multiangle laser light scattering (MALLS) detector from Wyatt, a Shodex OHpak SB-802 HQ column (linear, 8 mm  300 mm; pore size, 10 nm; bead size, 8 μm; exclusion limit, 4  103), and a Shodex OHpak SB-803 HQ column (linear, 8 mm  300 mm; pore size, 80 nm; bead size, 6 μm; exclusion limit, 1  105) in 0.02% NaN3 aqueous solution containing NaNO3 (0.1 M). The infrared (IR) spectra were recorded using a Perkin-Elmer Spectrum RXI FTIR spectrometer. The electrospray ionization time-of-flight high-resolution mass spectrometry (ESITOF HRMS) measurement was performed on a Micromass ZABSpec-Tof spectrometer by Centre R
egional de Mesures Physiques de l’Ouest, Universit
e de Rennes 1. The ultravioletvisible (UV-vis) spectra were measured with a 10 mm path length using a Jasco V-550 spectrophotometer, which used a deuterium lamp as the light source for the UV range (190350 nm) and a halogen lamp for the visible range (330900 nm), equipped with an EYELA NCB-1200 temperature controller. The elastic and dynamic light scattering experiments were carried out using an ALV setup. Transmission electron microscopy (TEM) experiments were carried out using a CM200 Philips microscope. Determination of Tcp. A typical method for the determination of the polymer’s Tcp is as follows: Mal7-b-PNIPAM220 solution in deionized water (2 g L-1) was cooled in an ice bath for 2 min, and then the resulting clear solution was transferred to a poly(methyl methacrylate) cell with 1 cm light path length. The transmittance at 500 nm of the aqueous solution was recorded on a UV-vis spectrophotometer with a temperature controller. The solution temperature was gradually raised at the heating rate of 1.0 °C min-1.

Static and Dynamic Light Scattering Measurements. Scattering measurements were performed using an ALV laser goniometer, which consists of a 22 mW HeNe linear polarized laser operating at a wavelength of 632.8 nm, an ALV-5004 multiple τ digital correlator with 125 ns initial sampling time, and a temperature controller. The accessible scattering angles range from 20° to 155°. The aqueous solutions of copolymers (typically 0.2 g L-1) were filtered directly into the glass cells through 0.45 μm MILLIPORE Millex LCR filter. In case of the temperature-dependent experiments, the sample temperature was changed stepwise as follows: the sample temperature was heated or cooled to the desired temperature and kept for 20 min prior to measurement. Data were collected using digital ALV Correlator Control software, and the counting time for measuring the elastic or the quasi-elastic scattering intensities varied for each sample from 180 to 300 s. The relaxation time distributions, A(t), were in the sequence obtained using CONTIN analysis of the autocorrelation function, C(q,t). Diffusion coefficients D were calculated from

Article following equation: Γ q2

¼D

ð1Þ

qf0

where Γ is relaxation frequency (Γ = τ-1) and q is the wave vector defined as following equation: q ¼

  4πn θ sin λ 2

ð2Þ

where λ is the wavelength of the incident laser beam (632.8 nm), θ is the scattering angle, and n is the refractive index of the media. Consequently, the hydrodynamic radius (Rh) was calculated from the Stokes-Einstein relation as follows: Rh ¼

kB T 2 kB T q ¼ 6πηΓ 6πηD

ð3Þ

where kB is the Boltzmann constant, T is the temperature, and η is the viscosity of the medium. As for the radius of gyration (Rg), it was calculated from the elastic part I(q) of the scattered intensity using the Guinier approximation as follows: ln I ¼ ln I0 -

1 2 2 q Rg 3

ð4Þ

where I is the scattering intensity and I0 is the scattering intensity at q = 0.

Transmission Electron Microscopy (TEM) Measurements. Transmission electron microscopy (TEM) images were recorded on a Kodak SO163 film using a CM200 Philips microscope operating at 80 kV. A typical method for preparing TEM samples is as follows: aqueous solution of a copolymer sample (0.2 g L-1) and a carbon-coated copper grid, which was rendered hydrophilic by glow discharge treatment, were kept in an oven at 90 °C. The copolymer solution (4 μL) was dropped on to the grid and then dried in the oven.

Results and Discussion Synthesis of Maltoheptaose-block-poly(N-isopropylacrylamide) (Mal7-b-PNIPAMn). We first synthesized an alkyne-functionalized maltoheptaose and azide-functionalized PNIPAMs to obtain hybrid maltoheptaose-block-PNIPAM copolymers by means of “click” chemistry. For introducing an alkyne group at the reducing end of maltoheptaose, we used a

Scheme 1. Strategy Used for the Syntheses of Mal7-block-PNIPAMn: (A) Synthesis of Maltoheptaose Having β-Configured N-Acetylpropargyl Group; (B) Syntheses of PNIPAMs with Azido End Groups by ATRP; (C) Block Coupling Reactions by “Click” Chemistry

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convenient method27 allowing access to N-propargyl glycosylamide from unprotected saccharide, as shown in Scheme 1. Maltoheptaose was suspended in neat propargylamine for 72 h, and the intermediate N-propargyl maltoheptaosylamine was subsequently converted into the corresponding stable β-N-acetylpropargyl maltoheptaosylamide (1) by selective N-acylation with acetic anhydride in methanol. The characteristic 1H NMR signals due to the anomeric proton at 5.5 and 5.0 ppm (J = 8.9 and 9.2 Hz), which were multiplied by the restricted rotations (rotamers) of the N-acetyl group, confirmed the β-anomeric configuration of 1 (see Supporting Information). PNIPAMs having terminal azido groups (N3-PNIPAMn) were synthesized by ATRP using azide-functionalized initiator (Scheme 1). In the 1H NMR spectrum of the resulting polymer, the signals assignable to the protons of the PNIPAM (Figure 1a-d) and the methylene protons adjacent to the azido group (Figure 1e,f) were observed. In addition, a characteristic signal due to the azido group was observed at 2104 cm-1 in the IR spectrum of the product (Figure 2). Thus, the obtained polymer was assignable to PNIPAM having a terminal azido group (N3-PNIPAM45). The results of the polymerizations are listed in Table 1. We obtained narrow distribution of N3-PNIPAMn having different degree of polymerization (DP) values by changing the monomer/initiator ratios in feeds. More detailed characterizations of the polymerizations are described in our previous report.26 We then performed “click” reaction in water between 1 and N3-PNIPAMn using sodium ascorbate and copper sulfate catalysts at room temperature for 24-48 h, as shown in Scheme 1.28,29 Here, we used an excess amount of compound 1, which was removed by dialysis after the reaction, to complete coupling reactions. After the complete reactions were confirmed with the disappearance of the signals due to azide groups around 2100 cm-1 by IR analysis, the mixtures were purified by dialysis in water and then freezedried to obtain all products as white solids. They were characterized by SEC and NMR analyses. Figure 2 shows the IR spectra of N3-PNIPAM45 and Mal7-b-PNIPAM45. The results showed that the signal due to the azide group in the spectrum of N3-PNIPAM45 at 2104 cm-1 has completely disappeared, indicating that there is no unreacted azide group in Mal7-b-PNIPAM45. The SEC trace of Mal7-b-PNIPAM45 as shown in Figure 3 displayed a unimodal signal, which clearly shifted toward a higher molar mass region as compared to the corresponding PNIPAM precursors. This indicated that Mal7-b-PNIPAMn had efficient conjugation of the saccharidic block onto the polymer chain. In the 1H NMR spectrum of Mal7-b-PNIPAM45, the characteristic signals due to the methine proton of the triazole ring (Figure 4g), the protons of PNIPAM group (Figure 4a-f), and the protons of the maltoheptaose group (Figure 4h,i) were observed. In addition, the quantitative coupling reaction between 1 and N3-PNIPAM45 was confirmed by comparing the integral ratio of the signals due to maltoheptaose (Figure 4i) and PNIPAM (Figure 4d). Therefore, we successfully synthesized targeted diblock copolymer containing PNIPAM and maltoheptaose moiety by the coupling method using “click” chemistry. The characteristics of N3-PNIPAMn and diblock copolymers are summarized in Table 1. The Tcp of both N3-PNIPAMn and diblock copolymers were shown to be correlated with the degree of polymerizations of the PNIPAM blocks; i.e., polymers with

longer NIPAM blocks showed lower Tcp values. In addition, each diblock copolymer showed higher Tcp values than those of the corresponding N3-PNIPAMn. This is in good agreement with the previous reports which indicated that the LCST of PNIPAM is dependent on both the molecular weight and hydrophilicity of the end group.26,30-32 Considering the potential applications of

(27) Halila, S.; Manguian, M.; Fort, S.; Cottaz, S.; Hamaide, T.; Fleury, E.; Driguez, H. Macromol. Chem. Phys. 2008, 209, 1282–1290. (28) We confirmed that no oxidative depolymerization reported in ref 29 had occurred under this reaction condition. (29) Uchida, K.; Kawakishi, S. Agric. Biol. Chem. 1986, 50, 2579–2583.

(30) Xia, Y.; Yin, X.; Burke, N. A. D.; St€over, H. D. H. Macromolecules 2005, 38, 5937–5943. (31) Xia, Y.; Burke, N. A. D.; St€over, H. D. H. Macromolecules 2006, 39, 2275–2283. (32) Kujawa, P.; Segui, F.; Shaban, S.; Diab, C.; Okada, Y.; Tanaka, F.; Winnik, F. M. Macromolecules 2006, 39, 341–348.

2328 DOI: 10.1021/la902743y

Figure 1. 1H NMR spectrum of N3-PNIPAM45 in CDCl3.

Figure 2. IR spectra of (A) N3-PNIPAM45 and (B) Mal7-b-PNIPAM45.

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Article Table 1. Molecular and Thermal Characteristics of N3-PNIPAMn and Diblock Copolymers N3-PNIPAMn

sample

Mn,th

Mn,NMRa

DPNMRa

diblock copolymers Mn,SECb

(Mw/Mn)

b

Tcpc

(°C)

sample

Tcpc (°C)

2900 3300 28 5700 (1.19) 51.5 Mal7-b-PNIPAM28 73.9 N3-PNIPAM28 5400 5300 45 10400 (1.20) 44.2 Mal7-b-PNIPAM45 55.7 N3-PNIPAM45 11800 13600 119 23700 (1.23) 38.6 Mal7-b-PNIPAM119 42.5 N3-PNIPAM119 21100 25100 220 34900 (1.23) 36.4 Mal7-b-PNIPAM220 39.4 N3-PNIPAM220 a Determined by 1H NMR spectra in CDCl3 at room temperature. b Determined by SEC in DMF containing 0.01 mol L-1 LiCl. c Determined by turbidimetry. Conditions: concentration = 2.0 g L-1, heating rate = 1.0 °C min-1, λ = 500 nm.

Figure 3. SEC traces of N3-PNIPAM45 and Mal7-b-PNIPAM45.

Figure 4. 1H NMR spectrum of Mal7-b-PNIPAM45 in D2O.

diblock copolymers for thermoresponsive drug encapsulation/ release system, the Tcp should be close to physiological temperature. Therefore, we hereafter focused on the self-assembling properties of Mal7-b-PNIPAM220 because its Tcp (39.4 °C) is lower than the other polymers and close to the physiologic temperature. Self-Assembled Nanoparticles Obtained from N3-PNIPAM220 and Mal7-b-PNIPAM220 Systems: Elastic and Dynamic Light Scattering Experiments. Scattering measurements of Mal7-b-PNIPAM220 and N3-PNIPAM220 aqueous solutions were carried out below and above the Tcp. Below the Tcp, scattering measurements showed typical behavior for single chain. For those molar masses, both systems are soluble in water at room temperature and the scattered intensities were very weak. Thus, the hydrodynamic radii (Rh) were only few nanometers, reflecting the dynamics of single Mal7-b-PNIPAM220 and N3-PNIPAM220 chains. Above the Tcp (47 °C), whereas the aqueous N3-PNIPAM220 system shows rather a large size and polydisperse Langmuir 2010, 26(4), 2325–2332

distribution, a single-exponential decay of the autocorrelation function, giving a very narrow relaxation-time distribution, and a q2 variation of relaxation frequency (Γ) were observed for Mal7-b-PNIPAM220, as illustrated in Figure 5. These results indicated that the presence of maltoheptaose linked to N3-PNIPAM220 to form Mal7-b-PNIPAM220 diblock copolymer is at the origin of this behavior and plays an important role in the size distribution of the formed nanoparticles in water. Thus, the Rh values of Mal7-b-PNIPAM220 (Rh = 144 nm) and the other nanoparticles discussed hereafter were calculated from the Stokes-Einstein relation (eq 3). One notes, however, a slight deviation of Γ vs q2 due to the form factor or, more precisely, to the size (Rh = 144 nm). Indeed, at this size, the product qRg > 1 for high angles in the light scattering, and we expect a slight deviation from q2 behavior. To gain more understanding on the thermoresponsiveness as well as the shape of the nanoparticles obtained in both systems, we investigated a temperature dependence of the elastic light scattering intensities and the Rh values of Mal7-b-PNIPAM220 and N3PNIPAM220. First, the samples were heated form 25 to 70 °C and then cooled to 25 °C. As shown in Figure 6, strong elastic scattering intensities of N3-PNIPAM220 and Mal7-b-PNIPAM220 were measured above their Tcps. This indicated the self-aggregation of PNIPAM chains due to their coil/globule transition around LCST. On the other hand, the dynamic light scattering results show that the measured Rh values of N3-PNIPAM220 increases as a function of the temperature to reach a maximum of about 350 nm at roughly 40 °C and then a decrease to about 200 nm at 67 °C. Such observations for the self-aggregation of PNIPAM have been intensively investigated by Wu and coworkers.33-35 The PNIPAM chains first aggregate into “molten” globule structure which consists of a dense core and a swollen shell by heating from room temperature to the LCST. Then, further heating shrinks the molten globule to fully collapsed globule structure. In addition, there exists a clear hysteresis of the Rh value in a heating-and-cooling cycle. The observed behavior of N3-PNIPAM220 is in good agreement with these reports. As for the Mal7-b-PNIPAM220 diblock copolymer, the measured Rh values increase also as a function of temperature to reach a maximum of about 200 nm at roughly 45 °C and then a slight decrease to about a plateau of about 150 nm at 67 °C. However, in sharp contrast to N3-PNIPAM220, the Rh value of Mal7-b-PNIPAM220 did not show a hysteresis in the heating-and-cooling cycle. This should indicate that the Mal7-b-PNIPAM220 formed a thermodynamically stable and reversible self-assembled nanoparticles due to its amphiphilic architecture where the hydrophilic maltohepthaose blocks act as a protection layer (shell) enhancing the stability of the formed self-assembled morphology. It should be noted that these Rh values are obtained by slowly increasing or decreasing the temperature with a heating rate of roughly 1 °C/min. (33) Wu, C.; Wang, X. Phys. Rev. Lett. 1998, 80, 4092–4094. (34) Wang, X.; Wu, C. Macromolecules 1999, 32, 4299–4301. (35) Cheng, H.; Shen, L.; Wu, C. Macromolecules 2006, 39, 2325–2329.

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Figure 5. (A) Dynamic light scattering autocorrelation function (circle) and relaxation-time distribution (solid line) of Mal7-b-PNIPAM220

in water (conditions: [Mal7-b-PNIPAM220] = 0.2 g L-1; scattering angle, θ = 90°; temperature, 47 °C). (B) Dependence of the relaxation frequency (Γ) on the square of the wave vector (q2).

Figure 6. Hydrodynamic radius (upper) and elastic scattering intensity (lower) as a function of temperature of (A) N3-PNIPAM220 and (B) Mal7-b-PNIPAM220 in water (conditions: [N3-PNIPAM220] and [Mal7-b-PNIPAM220] = 0.2 g L-1; scattering angle, θ = 90°).

We then investigated the shape of the nanoparticles of Mal7-bPNIPAM220 by calculating the ratio of the radius of gyration (Rg) to Rh. Here, the Rg value was determined from the slope of the Guinier plot calculated from the elastic light scattering intensity (eq 4). As shown in Figure 7, the Rg of Mal7-b-PNIPAM220 was found to be 145 nm, which leads to a ratio Rg/Rh = 1.01. It is interesting to note that when the temperature changed directly from room temperature to 47 °C, the Rh of Mal7-b-PNIPAM220 was calculated to be 144 nm. Different results could be obtained when the temperature is increasing slowly as discussed above (see Figure 6). Here, the theoretical values of Rg/Rh for solid spherical and vesicular particles are 0.775 and 1.0, respectively.36 Therefore, our result indicated that Mal7-b-PNIPAM220 forms vesicular particles in water above the Tcp. On the other hand, the Guinier plot of N3-PNIPAM220 showed two different slopes corresponding to Rg values of 233 and 122 nm. This indicates the presence of large aggregates and isolated small particles. Indeed, a general behavior of the system made of small and large particles (in this case aggregates) will have a scattering intensity that increase (36) Stauch, O.; Schubert, R.; Savin, G.; Burchard, W. Biomacromolecules 2002, 3, 565–578.

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dramatically at small q values and decreases rapidly at high q values. This results in the existence of two different slopes in a Guinier plot with the variation of ln I vs q2 as observed in this work. Transmission Electron Microscope (TEM): Analysis of the Self-Assembled Particle of Mal7-b-PNIPAM220. We then performed TEM measurements for better understanding and imaging of the self-assembled particles by comparing the results with the light scattering experiments. The samples used for TEM measurements were prepared in an oven at 90 °C to keep the temperatures of the samples higher than their Tcps. In the TEM image of Mal7-b-PNIPAM220, a clear vesicular morphology was observed as shown in Figure 8. On the other hand, polydisperse nanoparticles and large aggregates were observed in the TEM image for N3-PNIPAM220 (Figure 9). These TEM images clearly supported the results obtained from light scattering experiments. The presence of maltoheptaose block, even though it has seven glucopyranosyl units, affected significantly the self-assembly and the stability of Mal7-b-PNIPAM220 nanoparticles. Therefore, thermoresponsive amphiphilic Mal7-b-PNIPAM220 formed vesicular particles by the self-assembly in water at temperatures above Tcp. Additionally, the TEM image Langmuir 2010, 26(4), 2325–2332

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Figure 7. Guinier plots calculated from the elastic light scattering intensity of N3-PNIPAM220 (A) and Mal7-b-PNIPAM220 (B) in water (conditions: [N3-PNIPAM220] and [Mal7-b-PNIPAM220] = 0.2 g L-1; temperature, 47 °C).

Figure 8. TEM image of a dried dispersion of Mal7-b-PNIPAM220 prepared at 90 °C.

Figure 10. TEM image of a dried dispersion of Mal7-b-PNIPAM119 prepared at 90 °C.

(Figure 10). Here, the volume fractions of maltoheptaose blocks (φM) of Mal7-b-PNIPAM220 and Mal7-b-PNIPAM119 are 0.037 and 0.066.37-39 Such volume fractions indeed favor vesicular morphologies40 where PNIPAM is the hydrophobic part constituting the inside membrane and the malthoheptaose, the hydrophilic part, that constitutes the internal and external shell. A detailed description for encapsulation of hydrophilic and/or hydrophobic active molecules is under investigation and will be the subject of a forthcoming paper.

Conclusion

Figure 9. TEM image of a dried dispersion of N3-PNIPAM220 prepared at 90 °C.

of Mal7-b-PNIPAM119, whose PNIPAM (DP) is about half of Mal7-b-PNIPAM220, also showed the existence of vesicular morphology having very distinct contrast attributable to membranes (37) The volume fractions of maltoheptaose blocks of Mal7-b-PNIPAMn were calculated using the densities of amylose38 and PNIPAM (above LCST)39 reported in the literature (damylose = 1.36 g cm-3 and dPNIPAM = 1.13 g cm-3).

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A new class of hybrid block copolymers containing maltoheptaose and PNIPAM moieties, Mal7-b-PNIPAMn, was precisely synthesized by combining ATRP and “click” chemistry. Such hybrid copolymer systems, particularly Mal7-b-PNIPAM220 and Mal7-b-PNIPAM119, having very low volume fractions of maltoheptaose, self-assembled vesicular morphologies in water above their Tcps. The amphiphilic properties of the diblock copolymers arising from the thermoresponsive coil/globule conformational transition of the PNIPAM blocks along with the hydrophilic (38) Takahashi, Y.; Kumano, T.; Nishikawa, S. Macromolecules 2004, 37, 6827– 6832. (39) Collett, J.; Crawford, A.; Hatton, P. V.; Geoghegan, M.; Rimmer, S. J. R. Soc. Interface 2007, 4, 117–126. (40) Adams, D. J.; Butler, M. F.; Weaver, A. C. Langmuir 2006, 22, 4534–4540.

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maltoheptaose blocks was the driving force for the construction of vesicular morphologies. To the best of our knowledge, this is the first study on the thermoresponsive self-assembled morphology of oligosaccharide-based hybrid block copolymer. Further investigations on the effect of the volume fraction of PNIPAM and saccharidic blocks on the self-assembly as well as on the encapsulation and controlled release of active molecules are in progress and will be discussed in a forthcoming paper. Acknowledgment. This study was partly supported by a Grant-in-Aid for Japan Society for the Promotion of Science

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(JSPS) Fellows to I.O. and RTRA Nanoscience Project # FCSN2007-13P, Grenoble, to R.B. The authors thank Hayashibara Biochemical Laboratories, Inc., KOHJIN Co., and Mitsubishi Chemical Co. for providing chemical products. The authors also thank Dr. C. Rochas for his help, Ms. S. Boullanger for mass spectrometry, and Mr. P. Colin-Morel for SEC measurements. Supporting Information Available: 1H NMR and ESI-TOF HRMS spectra of 1. This material is available free of charge via the Internet at http://pubs.acs.org.

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