Thermoresponsive Self-Assemblies of Cyclic and Branched

Mar 23, 2012 - Elastic and quasi-elastic light scattering analysis of the dibock copolymers in H2O indicated that thermodynamic phase transitions of t...
1 downloads 8 Views 3MB Size
Download PDF
Article pubs.acs.org/Biomac

Thermoresponsive Self-Assemblies of Cyclic and Branched Oligosaccharide-block-poly(N-isopropylacrylamide) Diblock Copolymers into Nanoparticles Issei Otsuka,† Christophe Travelet,† Sami Halila,† Sébastien Fort,† Isabelle Pignot-Paintrand,† Atsushi Narumi,‡ and Redouane Borsali*,† †

Centre de Recherches sur les Macromolécules Végétales (CERMAV, UPR-CNRS 5301), affiliated with the Université Joseph Fourier (UJF) and member of the Institut de Chimie Moléculaire de Grenoble (ICMG, FR-CNRS 2607), BP53, 38041 Grenoble Cedex 9, France ‡ Department of Polymer Science and Engineering, Graduate School of Science and Engineering, Yamagata University, Jonan 4-3-16, Yonezawa 992-8510, Japan

ABSTRACT: This paper discusses the thermoresponsive nanoparticles obtained by self-assemblies of nonlinear oligosaccharidebased diblock copolymer systems. These diblock copolymers were synthesized by Cu(I)-catalyzed 1,3-dipolar azide/alkyne cycloaddition (“click” reaction) of propargyl-functionalized β-cyclodextrin (βCyD) and xyloglucooligosaccharide (XGO) with poly(N-isopropylacrylamide) (PNIPAM) having a terminal azido group prepared by atom transfer radical polymerization (ATRP). Elastic and quasi-elastic light scattering analysis of the dibock copolymers in H2O indicated that thermodynamic phase transitions of the PNIPAM blocks at their cloud points (Tcps ≈ 34 °C), around lower critical solution temperatures (LCSTs), triggered their self-assemblies into the nanoparticles. These nanoparticles had narrow size distributions and small interphases (i.e., sharp boundaries). The mean hydrodynamic radii (Rhs) of the βCyD and XGO-based nanoparticles were determined to be around 150 and 250 nm upon slow heating (i.e., step-by-step heating), and 364 and 91.5 nm upon fast heating, respectively, depending on a predominance of the interchain association or the intrachain contraction. Transmission electron microscope (TEM) and field emission gun-scanning electron microscopy (FEG-SEM) images of the nanoparticles clearly showed compact spherical nanoparticles whose cores are mainly made with the PNIPAM blocks, whereas the rough shells consist in the oligosaccharidic blocks.

■

INTRODUCTION Block copolymer self-assembly has been widely studied as one of the promising bottom-up strategies for providing a variety of functional nanomaterials,1−4 such as molecular delivery vehicles, cosmetics, patterning for nanoelectric devices, and so on. In particular, random walk or Gaussian “coil−coil” type block copolymers are well-known to self-assemble into diverse morphologies in solution (micelles, vesicles, and bilayers) and thin film state (spheres, cylinders, gyroids, and lamellas) from tens to hundreds of nm length scales.5 On the other hand, selfassemblies of block copolymers containing one or more rigid rod-like polymer blocks are little-known because the fundamental features of the rod-like polymer blocks (e.g., πconjugated polymers, polyisocyanates, aromatic polyesters, © 2012 American Chemical Society

polyamides, polyimines, helical proteins, etc.), such as chain topology, conformational entropy, molecular packing geometries, and scattering behaviors are different from the conventional coil-like polymer blocks and even specific for each rod-like block.6,7 Therefore, studies on fundamental phase behavior and self-assembly of the “rod−coil” type block copolymers are required both in solution and in bulk states. Block copolymers consisting of oligo- or polysaccharides as rod-like polymer blocks have been studied from the early 1980s and attract considerable attention because saccharides are Received: January 31, 2012 Revised: March 14, 2012 Published: March 23, 2012 1458

dx.doi.org/10.1021/bm300167e | Biomacromolecules 2012, 13, 1458−1465

Biomacromolecules

Article

Scheme 1. Synthesis of Oligosaccharide-block-PNIPAM Hybrid Diblock Copolymers

reasonable to design hybrid block copolymers containing stimuli-responsive polymers as one of the blocks. There have been, however, only a few reports on the saccharide-based hybrid block copolymers containing stimuli-responsive blocks.35,36 Recently, we have reported the first thermoresponsive self-assemblies of the hybrid diblock copolymers consisting of maltoheptaose (Mal7), a linear oligosaccharide composed of seven α-1,4-linked D-glucopyranosyl units, and PNIPAM blocks into vesicular nanoparticles (or polymersomes).36 In that study, thermoresponsive variation in hydrophobicity of the PNIPAM blocks around the LCST was the key for the self-assemblies of the hybrid block copolymers into vesicles. Therefore, the present study aims to further investigate thermoresponsive self-assemblies of new PNIPAMbased hybrid diblock copolymers containing nonlinear oligosaccharides. In this study, we report (1) synthesis of PNIPAM-based hybrid diblock copolymers consisting of cyclic βCyD and branched XGO as nonlinear oligosaccharidic blocks and (2) investigation of morphologies of their self-assembled nanoparticles in response to temperature. Here, βCyD is structurally defined as a cyclic Mal7, whereas XGO is a branched oligosaccharide consisting of a cellotetraose substituted with α-D-xylosyl residues and β-D-galactosyl-(1,2)-α-D-xylosyl residues. It is demonstrated that these nonlinear oligosaccharidic polymer blocks having similar molecular weights (i.e., 1,072 g mol−1 for βCyD and 1,360 g mol−1 for XGO) but different conformations (Scheme 1) lead to a difference in morphologies, and especially in sizes, of the nanoparticles obtained by thermoresponsive self-assemblies of the hybrid block copolymers.

abundant natural materials and have various biological activities; that is, saccharides are biocompatible and play important roles on cellular surfaces as recognition ligands for such as other cells, pathogens, and enzymes through a specific saccharide−lectin interaction.8,9 Some of these saccharidebased “hybrid” block copolymers consisting of oligo- or polysaccharides and synthetic polymer blocks express amphiphilic properties and self-assemble into nanoparticles such as micelles and vesicles (namely polymersomes) having shells of the saccharidic blocks and cores or bilayers of the synthetic blocks in aqueous media.10 In terms of the application of the self-assembled nanoparticles for active molecular delivery, such as drugs and genes, the shells of the nanoparticles are significant for stabilizing the structures and providing biocompatibility and targeting selectivity.4 Therefore, rigid rod-like structure, high water solubility, biocompatibility, and biorecognition property of the oligo- and polysaccharides make them attractive candidates for the component of the block copolymers, which can provide promising molecular delivery vehicles. While the oligosaccharides on cellular surfaces generally have intricate structures, most of the oligo- and polysaccharides in the hybrid block copolymers reported so far had linear structures. Thus, it is of great interest, both from physicochemical and biomedical perspectives, to prepare new hybrid block copolymers consisting of nonlinear saccharidic blocks, such as branched11,12 and cyclic13 oligosaccharides, and investigate their selfassemblies. As the synthetic blocks of the saccharide-based hybrid block copolymers, commonly used polymers (e.g., polystyrene,14−20 polyacrylates,21−23 poly(vinyl acetate),24 and polyamides25) and biocompatible polymers (e.g., polypeptides,26−28 poly(εcaprolactone),29,30 and poly(ethylene glycol)31−35) have been adopted so far. Although some of these hybrid block copolymers self-assemble into nanoparticles in selective solvents, these nanoparticles hardly vary their structures in response to external stimuli. Indeed, regulating self-assembly by external stimuli is very important for efficient molecular encapsulation and release in a controlled manner. Thus, it is

■

EXPERIMENTAL SECTION

Materials. Tamarind seed xyloglucan (purity 95.0%) was purchased from Dainippon Pharmaceutical Co., Ltd., Japan, and used as received. N-Isopropylacrylamide (NIPAM) was kindly supplied from KOHJIN Co., Japan. β-Cyclodextrin, sodium ascorbate, and copper sulfate were purchased from Sigma-Aldrich and used as

1459

dx.doi.org/10.1021/bm300167e | Biomacromolecules 2012, 13, 1458−1465

Biomacromolecules

Article

treatment, were kept in an oven at 60 °C. The copolymer suspension (4 μL) was dropped onto the grid, and then dried in the oven. FEG-SEM Observations. FEG-SEM images were obtained in secondary electron imaging mode using a Zeiss Ultra 55 FEG-SEM at an accelerating voltage of 5 kV using an in-lens detector. A typical method for preparing FEG-SEM samples is as follows: aqueous solution of a diblock copolymer sample (0.2 g L−1) and a mica sheet fixed on an aluminum stub were kept in an oven at 60 °C. The copolymer suspension (10 μL) was dropped onto the mica and then dried in the oven. The specimens were coated with 1 nm of electron beam evaporation platinum/carbon or 2 nm of carbon rods evaporation.

received. Water was purified by a Milli-Q water purification system (Billerica, MA, U.S.A.). Mono-6-N-propargylamino-6-deoxy-βCyD (propargyl-βCyD)37 and N-(XGO)-3-acetamido-1-propyne (propargyl-XGO)12 were synthesized according to previous reports. The synthesis of PNIPAM having a terminal azido group (N3-PNIPAM; Mn = 25100, Mw/Mn = 1.23) was reported elsewhere.38 Instruments. 1H NMR spectra were recorded using 400 MHz Bruker Avance DRX400. Size exclusion chromatography (SEC) was performed at 40 °C using a Agilent 390-MDS system (290-LC pump injector, ProStar 510 column oven, 390-MDS refractive index detector) equipped with Knauer Smartline UV detector 2500 and two Agilent PolyPore PL1113−6500 columns (linear, 7.5 × 300 mm; particle size, 5 μm; exclusion limit, 200−2000000) in DMF containing lithium chloride (0.01 M) at the flow rate of 1.0 mL min−1. Infrared (IR) spectra were recorded using a Perkin-Elmer Spectrum RXI FTIR spectrometer. Light scattering experiments were carried out using an ALV setup. Transmission electron microscopy (TEM) was carried out using a CM200 Philips microscope. Field emission gun-scanning electron microscopy (FEG-SEM) was carried out using a Zeiss Ultra 55 FEG-SEM microscope (CMTC-INP, Grenoble). Synthesis of βCyD-b-PNIPAM. To a solution of N3-PNIPAM (200 mg, 5.73 × 10−6 mol, 1 equiv), propargyl-βCyD (13.5 mg, 1.15 × 10−5 mol, 2 equiv), and sodium ascorbate (12.3 mg, 6.21× 10−5 mol, 11 equiv) in water (2.95 mL) was added a 0.01 M solution of CuSO4 (0.573 mL, 5.73 × 10−6 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 azido 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 βCyD-b-PNIPAM as a white solid (159 mg, 77%). Synthesis of XGO-b-PNIPAM. To a solution of N3-PNIPAM (200 mg, 5.73 × 10−6 mol, 1 equiv), propargyl-XGO (23.4 mg, 1.72 × 10−5 mol, 3 equiv), and sodium ascorbate (11.4 mg, 5.73× 10−5 mol, 10 equiv) in water (9.95 mL) was added a 0.01 M solution of CuSO4 (0.573 mL, 5.73 × 10−6 mol, 1 equiv). The mixture was stirred at room temperature for 63 h until the IR spectra showed complete disappearance of the signal due to the azido 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 XGO-b-PNIPAM as a white solid (195 mg, 94%). Light Scattering Measurements. Scattering measurements were performed using an ALV laser goniometer, which consists of a 22 mW HeNe linearly polarized laser operating at a wavelength of 632.8 nm, an ALV-5000/EPP multiple τ digital correlator with 125 ns initial sampling time, and a temperature controller. The accessible scattering angles (θs) range from 23 to 155°. The aqueous solutions of copolymers (typically 0.2 g L−1) were filtered directly into the glass cells through a 0.1 μm Whatman hydrophilic PTFE filter. In the case of the temperature-dependent experiments, the sample temperature was changed stepwise as follows: the sample temperature was heated up or cooled down to the desired temperature and kept at that temperature for 20 min prior to measurement to ensure that the system reached its equilibrium state (in terms of light scattering intensity and Rh value). Data were collected using digital ALV correlator control software and the counting time for measuring the elastic or the quasi-elastic scattering varied for each sample from 180 to 300 s. The reproducibility of the carried out measurements was checked at least two times. The relaxation-time (τ) distributions were obtained using the CONTIN analysis applied to the quasi-elastic light scattering autocorrelation functions (g(2)-1s).39 From the linear dependence of the relaxation frequency (1/τ) on the square of the wave vector (q2), the diffusion coefficient (Ddiff) of the nanoparticles was calculated. The Rh value was then obtained from Ddiff with the Stokes−Einstein relation. The detailed analysis of data was reported in the previous publications.36,40 TEM Observations. TEM images were recorded on a Kodak SO163 film using a CM200 Philips microscope operating at 120 kV. A typical method for preparing TEM samples is as follows: aqueous solution of a diblock copolymer sample (0.2 g L−1) and a carboncoated copper grid, which was rendered hydrophilic by glow discharge

■

RESULTS AND DISCUSSION Synthesis of Oligosaccharide-block-PNIPAM Hybrid Diblock Copolymers. The hybrid diblock copolymers containing βCyD (βCyD-b-PNIPAM) and XGO (XGO-bPNIPAM) were synthesized by click reactions of previously reported PNIPAM having a terminal azido group (N3PNIPAM; Mn = 25100, Mw/Mn = 1.23)36,38 with propargylfunctionalized βCyD (propargyl-βCyD)37 and XGO (propargyl-XGO).12 The “click” reactions were performed in water using sodium ascorbate and copper sulfate as the catalytic system at room temperature for a few days (Scheme 1). The reaction mixtures were purified by dialysis in H2O to remove the catalysts and excess alkynes and then freeze-dried to obtain products as white solids. The reactions were monitored by checking IR spectra, as shown in Figure 1. The signal corresponding to the azido group of N3-PNIPAM around 2100 cm−1 disappeared in the spectra of the products, indicating that the azido group of N3-PNIPAM was completely reacted with alkyne groups of propargyl-βCyD and propargylXGO. The obtained products were then characterized by 1H NMR analysis. Signals corresponding to the protons of PNIPAM blocks (Figure 2, signals “a−d” shown in green), the methine protons of triazole rings (Figure 2, signal “e” shown in red), and oligosaccharidic protons (Figure 2, signal “f” shown in blue) were observed in the spectra of the products. In addition, the SEC traces of the products shown in Figure 3 displayed slightly but clearly shifted signals toward higher molar mass region as compared to that of N3-PNIPAM, indicating that the products had efficient conjugation of the oligosaccharidic blocks to the PNIPAM block. Thus, the obtained products were assigned to the hybrid diblock copolymers containing PNIPAM and oligosaccharidic blocks. Thermoresponsive Self-Assemblies of the Hybrid Diblock Copolymers. Solubility of PNIPAM in water drastically changes depending on temperature, that is, PNIPAM dissolves in cold water but becomes insoluble when it is heated higher than the lower critical solution temperature (LCST ≈ 32 °C).41 Consequently, it is reasonable to speculate that the “double hydrophilic” hybrid diblock copolymers at room temperature become amphiphilic and self-assemble at higher temperature than their LCSTs. Indeed, we have recently demonstrated that the linear oligosaccharide based hybrid diblock copolymers (Mal7-b-PNIPAM) self-assembled into vesicular nanoparticles by heating above their Tcps ≈ 39 °C, while the N 3-PNIPAM homopolymer shrank to large aggregates in the same temperature.36 Thus, the thermoresponsive self-assembly behavior of βCyD-b-PNIPAM and XGOb-PNIPAM were characterized by static (elastic) and dynamic (quasi-elastic) light scattering analysis. Elastic light scattering intensities of aqueous solutions of the diblock copolymers (0.2 g L−1) were measured in a range of temperatures from 25 to 60 1460

dx.doi.org/10.1021/bm300167e | Biomacromolecules 2012, 13, 1458−1465

Biomacromolecules

Article

Figure 1. IR spectra of (A) N3-PNIPAM, (B) βCyD-b-PNIPAM, and (C) XGO-b-PNIPAM.

Figure 2. 1H NMR spectra of (A) βCyD-b-PNIPAM and (B) XGO-bPNIPAM in D2O.

°C (Figure 4). First, the samples were heated up step-by-step from 25 to 60 °C and then cooled down step-by-step to 25 °C at a heating/cooling rate of roughly 1 °C min−1. The light scattering intensities of both diblock copolymer suspensions observed at higher temperature than 34 °C were much stronger (by a factor of ca. 500) than those observed below 34 °C. This indicates that both diblock copolymers have their cloud points (Tcps) around 34 °C. Hydrodynamic radii (Rhs) of the diblock copolymers calculated by quasi-elastic light scattering measurements using the Stokes−Einstein relation36,40 showed the maximum values around their Tcps and then decreased to constant values (ca. 150 and 250 nm for βCyD-b-PNIPAM and XGO-b-PNIPAM, respectively) by heating above 50 °C. This should indicate that the changes in solubility of the PNIPAM blocks around the LCSTs triggered self-assemblies of the diblock copolymers into nanoparticles. It should be noted that the Rh values obtained at 25 °C for βCyD-b-PNIPAM (3.1 nm) and for XGO-b-PNIPAM (3.7 nm, data not shown) are comparable with the predicted characteristic radius of 3.6 nm for the single chains. Taking into account only the 25,100 g mol−1 N3-PNIPAM block, the radius of gyration (Rg) is given by the following equation when the system is in Θ-solvent conditions:

Figure 3. SEC traces of N3-PNIPAM (black solid line), βCyD-bPNIPAM (blue dotted line), and XGO-b-PNIPAM (red dash-dotted line).

R g = x 0.5b/60.5 = 2200.5 × 0.6/60.5 = 3.6 nm

where x is degree of polymerization of PNIPAM (x = 220) and b is typical characteristic length of the NIPAM monomer (i.e., 4× the length of the carbon−carbon single bond). In reality, the quality of the solvent can be lower than Θ-solvent, thus, the exponent given in the numerator (i.e., 0.5) in the above1461

dx.doi.org/10.1021/bm300167e | Biomacromolecules 2012, 13, 1458−1465

Biomacromolecules

Article

Figure 4. Hydrodynamic radius (upper) and elastic light scattering intensity (lower) as a function of temperature of (A) βCyD-b-PNIPAM and (B) XGO-b-PNIPAM in water (condition: [βCyD-b-PNIPAM] and [XGO-b-PNIPAM] = 0.2 g L−1; scattering angle, θ = 90°).

Figure 5. Quasi-elastic light scattering autocorrelation function (g(2)-1) and relaxation-time distribution of (A) βCyD-b-PNIPAM and (B) XGO-bPNIPAM (conditions: [βCyD-b-PNIPAM] and [XGO-b-PNIPAM] = 0.2 g L−1; scattering angle, θ = 50, 90, and 130°; temperature, 60 °C).

Figure 6. Dependence of the relaxation frequency (1/τ) on the square of the wave vector (q2) of (A) βCyD-b-PNIPAM and (B) XGO-b-PNIPAM (conditions: [βCyD-b-PNIPAM] and [XGO-b-PNIPAM] = 0.2 g L−1; scattering angle, θ = 25−155°; temperature, 60 °C).

mentioned equation should be smaller. Nevertheless, the

Quasi-elastic light scattering analysis was also carried out on both diblock copolymer systems at different angles at 60 °C (Figures 5 and 6). For this, the samples were plunged into the preheated thermoregulated bath and the measurements were

calculated characteristic radius is of the same order of magnitude as the experimentally obtained values. 1462

dx.doi.org/10.1021/bm300167e | Biomacromolecules 2012, 13, 1458−1465

Biomacromolecules

Article

Figure 7. (A) Dependence of the elastic light scattering intensity (I) on the wave vector (q) of βCyD-b-PNIPAM (conditions: [βCyD-b-PNIPAM] = 0.2 g L−1; scattering angle, θ = 23−140°; temperature, 60 °C) and (B) corresponding Guinier plot showing the fit in the Guinier region (qRg ≤ 1.3).47,48.

Figure 8. TEM images of the self-assemblies of (A) βCyD-b-PNIPAM and (B) XGO-b-PNIPAM.

systems.42,43 As for the elastic light scattering measurements of βCyD-b-PNIPAM at different angles at 60 °C (Figure 7A), the light scattering intensities exhibited a q−4-dependence in the highest q-value region and bent toward a plateau in the lowest q-value region. This curve represents the form factor of the particles in the considered q-range and corresponds to a Porod behavior.44 It indicates that the suspension is highly segregated and contains dense particles having small interphases (i.e., sharp boundaries) on the one hand and less dense solvent on the other hand. As for the elastic light scattering of XGO-bPNIPAM, such a reliable curve was not observed due to the fact that the nanoparticles are too small (Rh = 91.5 nm) for the accessible q-range (5.25 × 10−3 nm−1 ≤ q ≤ 2.48 × 10−2 nm−1). For βCyD-b-PNIPAM, the Guinier-plot45,46 of the data shown in Figure 7A leads to the radius of gyration Rg = 217 nm (Figure 7B). This Rg value is truly valid because it is calculated from the slope of the linear fit in the Guinier region, that is to say, for q-values in qRg ≤ 1.3.47,48 Moreover, at such a lower concentration (0.2 g L−1), the structure factor is almost equal to unity. Thus, the experimental ratio ρ = Rg/Rh = 0.60, giving a more precise idea on the morphology of the present nanoparticles, is in reasonable agreement with the value corresponding to hard spheres (ρ = 0.77).49 This result clearly explains the presence of the dense particles, leading to the Porod behavior, observed and discussed above.

carried out after the systems reached equilibrium states. At 60 °C, the self-assembled diblock copolymer systems showed single-exponential decays of quasi-elastic light scattering autocorrelation functions and very narrow relaxation-time distributions at low, medium, and high scattering angles (50°, 90°, and 130°) as shown in Figure 5. Consequently, the selfassembly conditions were well-controlled and the nanoparticles have monomodal and narrow size distributions. The linear dependences of the relaxation frequency (1/τ) on the square of the wave vector (q2) clearly indicate the Brownian diffusive motion of the nanoparticles (Figure 6). Here, the slopes are equal to the diffusion coefficients (Ddiffs) of the nanoparticles (1.43 × 10−8 cm2 s−1 and 5.68 × 10−8 cm2 s−1 for βCyD-bPNIPAM and XGO-b-PNIPAM, respectively), from which the Rh values were calculated with the Stokes−Einstein relation (364 and 91.5 nm for βCyD-b-PNIPAM and XGO-bPNIPAM, respectively). Comparatively, these Rh values notably differ from the ones obtained by the step-by-step heating procedure described above (ca. 150 and 250 nm for βCyD-bPNIPAM and XGO-b-PNIPAM, respectively). Thus, the slow heating (i.e., step-by-step heating) and the fast heating (i.e., plunging the samples into the preheated thermoregulated bath), privileging, respectively, interchain association and intrachain contraction, lead to self-assemblies having different characteristic sizes, as already reported elsewhere on other 1463

dx.doi.org/10.1021/bm300167e | Biomacromolecules 2012, 13, 1458−1465

Biomacromolecules

Article

Figure 9. FEG-SEM images of the self-assemblies of βCyD-b-PNIPAM obtained using in-lens detector ((A) wide view and (B) enlarged view).

Figure 10. FEG-SEM images of the self-assemblies of XGO-b-PNIPAM obtained using in-lens detector ((A) wide view and (B) enlarged view).

Microscopic Analysis of the Nanoparticles. Microscopic analysis of the block copolymer self-assemblies was performed to investigate morphologies of the nanoparticles. The samples were prepared by drop-casting aqueous suspensions of the diblock copolymers on substrates in an oven preheated at 60 °C (fast sample heating), temperature at which the diblock copolymer systems showed monodisperse and constant Rh values in the light scattering investigation. TEM images of βCyD-b-PNIPAM and XGO-b-PNIPAM showed spherical nanoparticles covered with “solar flare-like” shell structures as shown in Figure 8. Considering the PNIPAM blocks of the diblock copolymers are in a shrunken dehydrated state above LCSTs, these shell-like structures can be reasonably attributed to the hydrophilic blocks (i.e., βCyD and XGO), whereas the cores are mainly consisting of the PNIPAM blocks. Comparing the much larger sizes of the nanoparticles (hundreds nm) than the calculated Rh values of the single chains of the block copolymers (ca. 3−4 nm), some oligosaccharidic blocks should also get involved in the cores of the shrunken PNIPAM blocks. For both systems, although the sample preparation was carried out without staining, the cores appeared quite dark compared to the solar flare-like shells, meaning that the nanoparticles are compact as described above in the light scattering investigation. In addition, the rough surfaces of these nanoparticles due to oligosaccharidic blocks were clearly observed in FEG-SEM images of both βCyD-b-PNIPAM and XGO-b-PNIPAM systems as shown in Figures 9 and 10, respectively. The nanoparticle characteristic sizes obtained by quasi-elastic light scattering analysis, TEM, and FEG-SEM were in the same order of magnitude, except for the XGO-bPNIPAM system in which the nanoparticles spread a bit on substrates during drying (see, in particular, Figure 10), thus, leading to larger and rather polydisperse characteristic diameters in microscopy compared to light scattering. Therefore, these thermoresponsive self-assemblies were characterized as nanoparticles having cores of PNIPAM and shells of cyclic and branched oligosaccharides. It is important to note that the previously reported hybrid diblock copolymer systems, Mal7-b-

PNIPAM, self-assembled into vesicular nanoparticles although the weight fractions of Mal7 (f = 0.047), βCyD (f = 0.041), and XGO (f = 0.051) in the diblock copolymers are almost same. Thus, the difference in conformation between Mal7 (linear), βCyD (cyclic), and XGO (branched) strongly affected the selfassemblies of the diblock copolymers.

■

CONCLUSION The self-assemblies of nonlinear oligosaccharide-based diblock copolymers in response to temperature change were demonstrated by light scattering and microscopy techniques. The elastic light scattering measurements of the aqueous solutions of the diblock copolymers through step-by-step heating and cooling processes indicated that the phase transitions of PNIPAM blocks around 34 °C (Tcp) triggered the selfassemblies. By the rapid heating of the diblock copolymer systems at 60 °C, the diblock copolymers self-assembled into nanoparticles having narrow size distributions determined from the quasi-elastic light scattering analysis. The microscopy observations of the nanoparticles using TEM and FEG-SEM demonstrated the spherical-shape of the nanoparticles with cores mainly consisting of the collapsed PNIPAM blocks and the “solar flare-like” shells of the cyclic and branched oligosaccharidic blocks. Therefore, the self-assembly of the diblock copolymers consisting of nonlinear oligosaccharides as well as linear one36 and PNIPAM were successfully controlled by thermal stimulation. These systems should be evolved into other hybrid block copolymer systems which consist of complex-structured oligosaccharides having specific interactions with, for instance, lectins for targeted deliveries of active molecules.

■

AUTHOR INFORMATION

Corresponding Author

*Tel.: +33 (0)476 037 640. Fax: +33 (0)476 037 629. E-mail: [email protected] Notes

The authors declare no competing financial interest. 1464

dx.doi.org/10.1021/bm300167e | Biomacromolecules 2012, 13, 1458−1465

Biomacromolecules

■

Article

(35) Hernandez, O. S.; Soliman, G. M.; Winnik, F. M. Polymer 2007, 48, 921−930. (36) Otsuka, I.; Fuchise, K.; Halila, S.; Fort, S.; Aissou, K.; PignotPaintrand, I.; Chen, Y.; Narumi, A.; Kakuchi, T.; Borsali, R. Langmuir 2010, 26, 2325−2332. (37) Guoa, Z.; Jin, Y.; Lianga, T.; Liua, Y.; Xua, Q.; Liang, X.; Lei, A. J. Chromatogr., A 2009, 1216, 257−263. (38) 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. (39) Provencher, S. W. Makromol. Chem. 1979, 180, 201−209. (40) Dal Bó, A. G.; Soldi, V.; Giacomelli, F. C.; Travelet, C.; Jean, B.; Pignot-Paintrand, I.; Borsali, R.; Fort, S. Langmuir 2012, 28, 1418− 1426. (41) Fujishige, S.; Kubota, K.; Ando, I. J. Phys. Chem. 1989, 93, 3311−3313. (42) Zhang, G.; Wu, C. Adv. Polym. Sci. 2006, 195, 101−176. (43) Boyko, V.; Richter, S.; Pich, A.; Arndt, K.-F. Colloid Polym. Sci. 2003, 282, 127−132. (44) Teixeira, J. J. Appl. Crystallogr. 1988, 21, 781−785. See also: Bale, H. D.; Schmidt, P. W. Phys. Rev. Lett. 1984, 53, 596−599. (45) Guinier, A.; Fournet, G. Small Angle Scattering of X-rays; Wiley: New York, 1955. (46) Burchard, W. Adv. Polym. Sci. 1983, 48, 1−124. (47) Hamill, A. C.; Wang, S.-C.; Lee, C. T. Biochemistry 2007, 46, 7694−7705. (48) Hamill, A. C. Photocontrol of Protein Conformation through the Use of Photoresponsive Surfactants, Investigated by Small Angle Neutron Scattering. Ph.D. Dissertation, University of Southern California, CA, 2008; Dissertation available on the website http:// digitallibrary.usc.edu/assetserver/controller/item/etd-Hamill20080306.pdf. (49) Burchard, W. Polysaccharides: Structural Diversity and Functional Versatility; Dekker: New York, 2005.

ACKNOWLEDGMENTS This study was partly supported by the Ministère des Affaires Etrangères et Européennes (MAEE) − Japan Society for the Promotion of Science (JSPS) joint project (SAKURA program). NIPAM was kindly provided by KOHJIN Co. The authors thank Dr. K. Fuchise, Ms. P. Chaud, and Ms. Y. Sakai for their help in the synthesis.

■

REFERENCES

(1) Kim, J. K.; Yang, S. Y.; Lee, Y.; Kim, Y. Prog. Polym. Sci. 2010, 35, 1325−1349. (2) Kim, H.-C.; Park, S.-M.; Hinsberg, W. D. Chem. Rev. 2010, 110, 146−177. (3) Orilall, M. C.; Wiesner, U. Chem. Soc. Rev. 2011, 40, 520−535. (4) Tyrrell, Z. L.; Shen, Y.; Radosz, M. Prog. Polym. Sci. 2010, 35, 1128−1143. (5) Bates, F. S.; Fredrickson, G. H. Phys. Today 1999, 52, 32−38. (6) Olsen, B. D.; Segalman, R. A. Mater. Sci. Eng., R 2008, 62, 37−66. (7) Wang, Q. Soft Matter 2011, 7, 3711−3716. (8) Lee, Y. C.; Lee, R. T. Acc. Chem. Res. 1995, 28, 321−327. (9) Dwek, R. A. Chem. Rev. 1996, 96, 683−720. (10) Schatz, C.; Lecommandoux, S. Macromol. Rapid Commun. 2010, 31, 1664−1684. (11) Lohmann, J.; Houga, C.; Driguez, H.; Wilson, J.; Destarac, M.; Fort, S.; Le Meins, J.-F.; Borsali, R.; Taton, D.; Gnanou, Y. ACS Symp. Ser. 2009, 1023, 231−240. (12) Halila, S.; Manguian, M.; Fort, S.; Cottaz, S.; Hamaide, T.; Fleury, E.; Driguez, H. Macromol. Chem. Phys. 2008, 209, 1282−1290. (13) Kakuchi, T.; Narumi, A.; Miura, Y.; Matsuya, S.; Sugimoto, N.; Satoh, T.; Kaga, H. Macromolecules 2003, 36, 3909−3913. (14) Loos, K.; Stadler, R. Macromolecules 1997, 30, 7641−7643. (15) Loos, K.; Müller, A. H. E. Biomacromolecules 2002, 3, 368−373. (16) Yagi, S.; Kasuya, N.; Fukuda, K. Polym. J. 2010, 42, 342−348. (17) Mezger, T.; Cantow, H.-J. Makromol. Chem., Rapid Commun. 1983, 4, 313−320. (18) Kamitakahara, H.; Enomoto, Y.; Hasegawa, C.; Nakatsubo, F. Cellulose 2005, 12, 527−541. (19) Houga, C.; Le Meins, J.-F.; Borsali, R.; Taton, D.; Gnanou, Y. Chem. Commun. 2007, 3063−3065. (20) Bosker, W. T. E.; Agoston, K.; Cohen Stuart, M. A.; Norde, W.; Timmermans, J. W.; Slaghek, T. M. Macromolecules 2003, 36, 1982− 1987. (21) Haddleton, D. M.; Ohno, K. Biomacromolecules 2000, 1, 152− 156. (22) Chauvierre, C; Labarre, D.; Couvreur, P.; Vauthier, C. Macromolecules 2003, 36, 6018−6027. (23) Bertholon, I.; Lesieur, S.; Labarre, D.; Besnard, M.; Vauthier, C. Macromolecules 2006, 39, 3559−3567. (24) Bernard, J.; Save, M.; Arathoon, B.; Charleux, B. J. Polym. Sci., Part A: Polym. Chem. 2008, 46, 2845−2857. (25) Hashimoto, K.; Imanishi, S.; Okada, M.; Sumitomo, H. J. Polym. Sci., Part A: Polym. Chem. 1992, 30, 211−220. (26) Schatz, C.; Louguet, S.; Le Meins, J.-F.; Lecommandoux, S. Angew. Chem., Int. Ed. 2009, 48, 2572−2575. (27) Upadhyay, K. K.; Le Meins, J.-F.; Misra, A.; Voisin, P.; Bouchaud, V.; Ibarboure, E.; Schatz, C.; Lecommandoux, S. Biomacromolecules 2009, 10, 2802−2808. (28) Douy, A.; Gallot, B. Makromol. Chem. 1977, 178, 1595−1599. (29) Liu, J.-Y.; Zhang, L.-M. Carbohydr. Polym. 2007, 69, 196−201. (30) Li, B.-G.; Zhang, L.-M. Carbohydr. Polym. 2008, 74, 390−395. (31) Ziegast, G.; Pfannemüller, B. Carbohydr. Res. 1987, 160, 185− 204. (32) Pfannemüller, B.; Schmidt, M.; Ziegast, G.; Matsuo, K. Macromolecules 1984, 17, 710−716. (33) Akiyoshi, K.; Kohara, M.; Ito, K.; Kitamura, S.; Sunamoto, J. Macromol. Rapid Commun. 1999, 20, 112−115. (34) Ganji, F.; Abdekhodaie, M. J. Carbohydr. Polym. 2008, 74, 435− 441. 1465

dx.doi.org/10.1021/bm300167e | Biomacromolecules 2012, 13, 1458−1465