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Notes Aqueous Self-Assembly of Polystyrene Chains End-Functionalized with β-Cyclodextrin Cristiano Giacomelli,†,# Vanessa Schmidt,†,# Jean-Luc Putaux,§ Atsushi Narumi,⊥ Toyoji Kakuchi,*,‡ and Redouane Borsali*,§ Laboratoire de Chimie des Polyme`res Organiques, ENSCPB, Universite´ Bordeaux 1, 16 Av. Pey Berland, 33607 Pessac Cedex, France, Centro de Cieˆncias Exatas e Tecnologia (CCET), Universidade de Caxias do Sul (UCS), Rua Francisco Getu´lio Vargas 1130, 95070-560, Caxias do Sul, Brazil, Department of Polymer Science and Engineering, Graduate School of Science and Engineering, Yamagata University, Jonan 4-3-16, Yonezawa 992-8510, Japan, Division of Molecular Chemistry, Graduate School of Engineering, Hokkaido University, 060-8628, Sapporo, Japan, and Centre de Recherches sur les Macromole´cules Ve´ge´tales (CERMAV-CNRS), BP 53, F-38041 Grenoble Cedex 9, France Received November 7, 2008 Revised Manuscript Received December 15, 2008
Introduction Among the contemporary challenges in polymer science are, unquestionably, the development of novel functional nanomaterials using renewable natural resources from biomass, and the understanding of their structures and properties.1 The strategies undertaken in such an endeavor depend very much on the clever application of the current interdisciplinary knowledge. In many cases, breakthrough results have indeed been achieved by mimicking, in simpler ways, the behavior observed in nature. In polymer chemistry, this motivation alongside the advances in polymerization techniques has inspired the synthesis of a relatively recent class of polymeric systems containing pendant saccharide groups, and featuring a wide range of controlled architectures and functionalities.2,3 The high biocompatibility as well as the excellent solubility in water of various glycoconjugated macromolecules make them ideal candidates for the preparation of novel sugar-based hybrid materials, such as nonionic polymeric nanoparticles, surfactants, surface modifiers, and hydrogels.2 Cyclodextrins (CDs) are a series of R-1,4-linked cyclic oligosaccharides composed of 6, 7, or 8 D-(+)-glucose repeat units (corresponding to R-, β-, and γ-CDs, respectively). These molecules are usually toroidal-shaped with larger and smaller openings exposing primary and secondary hydroxyl groups to hydrophilic solvents. The inner cavity is, in contrast, hydrophobic and can act as guest-selective host (molecular recognition), thus forming supramolecular inclusion complexes. In general, these are stabilized by noncovalent interactions such * To whom correspondence should be addressed. E-mail: borsali@ cermav.cnrs.fr (R.B);
[email protected] (T.K.). † LCPO-ENSCPB-UBX 1. # CCET-UCS. ⊥ Yamagata University. ‡ Hokkaido University. § CERMAV (affiliated with Universite´ Joseph Fourier and member of the Institut de Chimie Mole´culaire de Grenoble).
as hydrogen bonding, van der Waals forces, electrostatic and dipole-dipole interactions, and steric effects.4-6 The adequate combination of such a distinctive feature of CDs with hydrophobic polymer chains (or other low molecular weight moieties)7 is of great interest in both academy and industry, because this can ultimately lead to novel polymer-CD amphiphiles capable of forming self-assembled structures in aqueous media (e.g., spherical micelles, vesicles, etc.).8-10 Such structures exhibit at the same time a tunable hydrophobic compartment that can serve as cargo space, and a hydrophilic shell that can participate in supramolecular recognition processes. Therefore, besides acting in the colloidal stabilization of the aggregates, the CD oligosaccharides located at the surface can operate as channels, through which only species that fit into the CD cavity (size specificity) can travel in and out of the nanoobjects (by analogy with the formation of polypseudorotaxanes),11 thus allowing for selective encapsulation and/or release of hydrophobic guest molecules. The hydrophobic nanoreservoir is presumably able to stabilize otherwise water-insoluble species that passed through the CD hydrophobic cavity. The efficiency of such a process and therefore the encapsulated quantity is dictated by the Flory-Huggins interaction parameter (χ), which describes the compatibility between the core polymer and the molecules being encapsulated.12-14 Hence, the choice of the polymer connected to the CD is a very important parameter in the design of such functional nano-objects. In the light of these considerations, there is consequently a huge interest in obtaining well-defined, stable polymer-βCD self-assemblies in water. Within this context, we have devoted considerable efforts to the synthesis of well-defined copolymers containing β-cyclodextrin (βCD) residues.15,16 Polystyrene (PS) chains end-functionalized with βCD (PS-βCD),15 and sevenarm PS with βCD core ((PS)7-βCD)16 were previously obtained using nitroxide-mediated controlled radical polymerization (NMRP).17 The as-synthesized systems were usually soluble in DMF and pyridine, while self-organization into reverse-type polymeric micelles consisting of βCD core surrounded by PS shell was observed in toluene, chloroform and THF upon direct dissolution.15,16 In spite of the amphiphilic nature of PS-βCD chains, their low solubility in water prevented the self-assembly to occur directly in water. Indeed, when the copolymer solubility in water is too low, as is the case of PS-βCD and (PS)7-βCD, clever manipulation strategies involving indirect dissolution methods are required to obtain assembled structures in water.12,18 In the present work, we report on the successful preparation of original nanoparticles consisting of a dense hydrophobic PS core stabilized by a thin hydrophilic βCD shell. These novel particles possess not only the capacity to recognize specific types of compounds through the well-known βCD functionality, but also and the ability to encapsulate hydrophobic species within the tunable synthetic polymer-based core.
Experimental Section Materials. PS38-βCD polymer was synthesized by nitroxide-mediated controlled radical polymerization (NMRP) technique using a singlesite 2,2,6,6-tetramethylpiperidinyloxy (TEMPO)-substituted βCD initia-
10.1021/bm801282d CCC: $40.75 2009 American Chemical Society Published on Web 01/16/2009
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Scheme 1. Chemical Structure (A) and Solution Behavior (B) of Polystyrene Chains End-Functionalized with β-Cyclodextrin (PS-βCD)
tor as described in a previous work (macromolecular characteristics: Mn (SEC) ) 5000 g/mol; Mw/Mn ) 1.11; [R]D23 ) +35.7° (c 1.0, CHCl3); degree of polymerization of PS block ) 38; volume fraction of hydrophobic PS block φPS ) 0.62).15 All other chemicals were of the highest purity available from Sigma-Aldrich, and used without any further purification. Preparation and Properties of Self-Assembled Structures. Preparation of Micellar Solutions. PS38-βCD nanostructures were prepared using the dialysis (or indirect dissolution) method. Typically, 3.5 mg of polymer were dissolved in 3.5 mL of DMF (a good solvent for both blocks) in a closed vial. The solution was allowed to stir for at least 3 h, and micellization was subsequently induced by slow (∼6.0 mL/h) addition of water up to 80% w/w DMF/water. Finally, the copolymer/organic solvent/water mixture was extensively dialyzed against pure water (dialysis bag MWCO ) 25000 g/mol; dialysis time ) 5 days, water was replaced periodically) to remove the organic solvent. Static and Dynamic Light Scattering (SDLS). SDLS 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 and an ALV-5000/EPP multiple τ digital correlator with 125 ns initial sampling time. Data were collected at 25.0 ( 0.1 °C using ALV Correlator Control software and the counting time was 900 s. In the sequence, the relaxation time distributions, A(t) were obtained using CONTIN analysis19 of the autocorrelation function, C(q,t). The relaxation frequency, Γ (Γ ) τ-1), generally depends on the scattering angle, and in the case of a diffusive particle, this frequency is q2-dependent.20 Consequently, the apparent diffusion coefficient (Dapp) at a given copolymer concentration (Cp) is calculated from Γ/q2|qf0 ) Dapp, where q is the wave vector defined as q ) 4πn/λ sin(θ/2), and λ is the wavelength of the incident laser beam, and θ is the scattering angle. The hydrodynamic radius (RH) (or diameter, 2RH) is calculated from the Stokes-Einstein relation RH ) kBT/6πηDapp, where kB is the Boltzmann constant, T is the temperature of the sample, and η is the viscosity of the medium. The deduction of micellar shape from angular dissymmetry measurements of scattered light intensity is described in details elsewhere.21 Transmission Electron Microscopy (TEM). TEM images were recorded using a CM 120 Philips microscope operating at 120 kV, and
equipped with a USC1000-SSCCD 2k × 2k Gatan camera. To prepare the TEM samples, 5 µL of an aqueous solution of particles was dropped onto a carbon-coated copper grid, which was rendered hydrophilic by UV/ozone treatment. Excess of micellar solution was gently removed using absorbent paper. Samples were then negatively stained with uranyl acetate.
Results and Discussion PS38-βCD polymer chains (Scheme 1A) were recently found to undergo self-organization into well-defined objects upon very slow addition of water to a dilute organic presolution in DMF, followed by extensive dialysis (Scheme 1B). Preliminary solubility tests with various polar organic solvents straightforwardly indicated that DMF was the best choice for this system, since it is a thermodynamically good solvent for both blocks, and miscible with water. The PS38-βCD copolymer dissolved molecularly in DMF as judged from dynamic light scattering (DLS) measurements, which revealed the presence of a fast dominant mode related to small scattering particles in solution (unimers) with a typical hydrodynamic radius (RH) of ∼3.0 nm (Figure 1). This size is fairly consistent with theoretical calculations based on the average unperturbed end-to-end distance (〈r〉 ) l · N1/2, where l is the length of one repeat unit and N is the number of repeating units)22 of a PS38 chain (〈r〉 ∼ 1.9 nm), connected to a βCD residue (〈r〉 ∼ 0.8 nm). The normalized scattered light intensity (IscN ) Isc/I0 (incident)) recorded in such an organic medium was, as expected, very low (IscN ∼ 0.10), thus corroborating the molecular dissolution of PS38-βCD chains in DMF within the concentration range studied in this work (Cporg ) 0.10 - 10.0 mg/mL). In addition, IscN depended linearly on Cporg, which is characteristic of dilute regimes. The slow relaxation mode in Figure 1 is due to irregular aggregates formed by multiple polymer chains interacting dynamically. The dynamic nature of such interactions was revealed by the fact that large particles were still detected immediately after high speed centrifugation or filtration with a 0.1 µm (100 nm) pore size membrane. Based on the physical chemical properties of each
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segment, it is reasonable to consider that interactions take place preferentially via styrene/βCD pairs, because the inner cavity of βCD is hydrophobic and can form supramolecular inclusion complexes with styrene-like molecules.23-25 However, styrene/ styrene and βCD/βCD interactions cannot be ruled out. It is also interesting to note that the scattered light intensity strongly depends on particle mass and size, implying that LS reports an intensityaverage size (i.e., in a medium containing essentially small particles, the presence of only a few large aggregates can still be detected).26 The addition of water at controlled rate and amount to the copolymer organic phase was then performed in order to induce the self-organization. During this process, the solvent quality gradually changes toward opposite directions for each block, becoming increasingly good for one block and poor for the other. At the critical water concentration (cwc), which primarily depends on the length of the hydrophobic block and the copolymer concentration (Cp), the micellization occurs to minimize the contact between the solvophobic block and the solvent.12,22,27 As a result, self-assembled objects were generated in solution. Finally, the copolymer/organic solvent/water mixture was extensively dialyzed against pure water (dialysis bag MWCO ) 25000 g/mol; dialysis time ) 5 days, water was replaced periodically) to remove the organic solvent. Though apparently simple, this preparation technique involves a considerable number of experimental variables that eventually play a decisive role on the self-assembling process. After a series of experiments dedicated to the optimization of the micelle preparation method for PS38-βCD system, it was established that good control over the polymer concentration in the organic phase (DMF), prior to the addition of water (Cporg in the presolution), the rate of water addition (rH2O), and the final water content before the dialysis (wH2O) were parameters of utmost relevance for the successful preparation of micellar nanoparticles with narrow size distribution profiles by DLS. For instance, transparent (clear) aqueous solutions that remained stable over periods of months were only obtained from presolutions with Cporg e 1.0 mg/mL, to which water was added at rH2O e 6.0 mL/h up to wH2O < 80% w/w. Otherwise, the solutions were typically opaque and material deposition (precipitation) took place within periods of a few hours. Such a behavior is usually observed in micellar systems prepared by phase inversion, which can give aggregation followed by precipitation if micelles (or aggregates) are already present in the organic phase. However, micellar nanoparticles do not exist in organic medium before the addition
Figure 1. Autocorrelation functions C(q,t) measured at scattering angle of 90° and respective distribution of the relaxation times, A(t), as revealed by CONTIN analysis for 1.0 mg/mL PS38-βCD solution in DMF.
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Figure 2. Variation of the normalized scattered light intensity (IscN) as a function of water increments during the preparation of a PS38βCD micellar solution using Cporg ) 1.0 mg/mL, rH2O ) 6.0 mL/h up to wH2O ) 75% w/w, followed by extensive dialysis against pure water.
of water according to light scattering experiments (very sensitive to particles in solution; Figure 1). We believe, therefore, that the pre-existing dynamical aggregates (styrene/βCD interactions) are at the origin of this phenomenon, since they can hinder chain mobility during self-assembling. This situation evolves upon increasing the initial concentration because the number of aggregates becomes progressively larger, ultimately leading to precipitation for Cporg > 1.0 mg/mL. The morphology of the objects originated at this point (onset of the micellization) might not necessarily be in thermodynamic equilibrium. The size and shape (including particle merging into larger irregular structures) of the aggregated may evolve not only as a function of the time, but also the water content.12,28,29 The interplay between these aspects finally dictates the size and stability of the self-assembled structures.28,29 On the basis of these experimental observations, the preparation of PS38-βCD assemblies in water was performed using Cporg ) 1.0 mg/mL, rH2O ) 6.0 mL/h and wH2O ) 75% w/w, followed by extensive dialysis against pure water. The first step of this micelle preparation protocol (micellization induced by water) was probed by light scattering experiments as shown in Figure 2. The very low normalized scattered light intensity in pure organic medium (IscN ∼ 0.10) reflected the molecular dissolution of individual polymer chains (unimers in DMF). Upon the addition of water to such a solution, the solvent quality changed progressively in opposite directions for each block, since water is a selective solvent for βCD and a precipitant for PS. At wH2O ) 13% w/w, an abrupt increase in the IscN-values due to selfassembly of unimers into larger scattering particles was observed, thus characterizing the onset of micellization which continued up to wH2O ) 26% w/w. Subsequently, the average IscN values remained practically constant until wH2O ) 75% w/w. At this point, the water addition step was completed, and resulting micellar aqueous/organic solution was then extensively dialyzed against pure water in order to ensure the removal of the organic phase. The latter can swell the micelle core and act as a plasticizer since it is a good solvent for PS. During the dialysis process, however, the solvent content of the core drops progressively with a consequent shrinking. Indeed, the hydrodynamic radius (RH) was found to decrease rapidly from ∼60
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Figure 4. TEM images of PS38-βCD self-assembled nanoparticles obtained in aqueous medium and negatively stained with uranyl acetate. Scale bar ) 200 nm.
Figure 3. Autocorrelation functions C(q,t) measured at scattering angles between 50 and 130° and distributions of the relaxation times A(t) at 90°, as revealed by CONTIN analysis plot for 0.11 mg/mL PS38βCD solution in water (A) with corresponding angular dissymmetry measurements (B). Top inset: respective q2 dependency of the relaxation frequency.
nm down to ∼40 nm within the first 2 days of dialysis. The virtually constant values observed afterward (from 2 to 5 days) suggested that all the organic phase was effectively removed from the medium (Figure S1). The resulting particles exhibited narrow distributions of relaxation times (µ2/Γ2 ) 0.10 - 0.18, by cumulants analysis) in DLS measurements (Figure 3A), with a single dominant relaxation mode corresponding to their diffusive motion in solution, which was confirmed by the q2 dependency of relaxation frequency20,26 (Figure 3A, inset). Therefore, the hydrodynamic radius (RH) of the particles could be unequivocally determined using the Stokes-Einstein relation (RH ) kBT/ 6πηDapp, where kB is the Boltzmann constant, T is the temperature of the sample, and η is the viscosity of the medium). The characteristic RH value of PS38-βCD spherical micellar nanoassemblies was 38 nm. The IscN recorded in this case was remarkably higher (IscN (water) ∼ 2.25; IscN (DMF) ∼ 0.10; roughly corresponding to a 20-fold increase, not taking into account differences arising from the different scattering media) due to the denser structure and larger size of the newly formed scattering particles as compared to the molecularly dissolved unimers.
The formation of dense micellar nanoparticles in the present case was corroborated by the dimensionless ratio F ) Rg/RH, which is a relation useful to characterize the shape of molecular aggregates. The Rg value of the particles previously described was 31 nm, as determined from dissymmetry measurements21,30 of the scattered light intensity (Figure 3B). Therefore, F ) 0.82 for PS38-βCD particles, thus indicating the formation of spherical homogeneous micelles (F ) 0.779 for homogeneous hard spheres).20 The morphology was further confirmed by TEM images, which revealed the formation of moderately polydisperse dense nanoparticles (Figure 4) whose radius varied from 35 to 100 nm (number average radius R ) 60 nm measured from 200 particles). It is worth noting, however, that particles may flatten to some extent on the carbon film, resulting in an overestimation of their projected radius. This effect may be counterbalanced if dehydration of the structure is significant during drying. Both effects are difficult to evaluate separately from TEM images. In spite of this, the typical size observed by both techniques (DLS and TEM) is larger than expected for well-defined elementary core-corona spherical micelles with fully stretched PS-βCD chains (∼12 nm). Such an observation is most probably related to the formation of clusters, as reported previously for other systems.31 In the present case, this phenomena is favored by interaction of styrene repeat units with βCD.23-25 High resolution cryo-TEM and X-rays scattering experiments will be carried out to investigate the existence of inner structural order9 in these particles in aqueous environment.
Conclusion Self-assembled nanoparticles consisting of a dense hydrophobic PS-based core stabilized by a thin hydrophilic βCD shell have been successfully prepared in aqueous medium using indirect dissolution methods applied to PS chains end-functionalized with βCD (a hybrid synthetic-natural copolymer system) for the first time to the best of our knowledge. In principle, these particles exhibit two distinctive and very important properties, that is, the ability to recognize specific types of compounds through the βCD functionality and the ability to encapsulate hydrophobic species within the PS-based core. On the basis of the current knowledge on macromolecular synthesis and keeping in mind the convenient synthetic strategy employed to prepare the system investigated herein, the hydrophobic block
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can be easily tailored so as to optimize the loading/cargo capacity of this kind of particulate system. Acknowledgment. R.B. acknowledges financial support from the CNRS, Universite´ Bordeaux 1, and Re´gion Aquitaine. C.G. and V.S. thank CAPES and CNPq, respectively. The authors are grateful to Dr. A. R. Brisson and Dr. J. Lai-Kee-Him for helpful discussions on electron microscopy images. Supporting Information Available. Dynamic light scattering measurements of micellar size as a function of dialysis time. This material is available free of charge via the Internet at http:// pubs.acs.org.
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BM801282D
