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Biomacromolecules 2009, 10, 32–40
Micelles and Polymersomes Obtained by Self-Assembly of Dextran and Polystyrene Based Block Copolymers Cle´ment Houga,† Joanna Giermanska,‡ Se´bastien Lecommandoux,† Redouane Borsali,§ Daniel Taton,† Yves Gnanou,*,† and Jean-Franc¸ois Le Meins*,† Universite´ de Bordeaux, Laboratoire de Chimie des Polyme`res Organiques UMR5629, ENSCPB-CNRS, 16 avenue Pey Berland, 33607, Pessac cedex, France, Universite´ de Bordeaux, Centre de Recherche Paul Pascal CNRS-UPR 8641, Avenue Albert Schweitzer, 33600 Pessac, France, and Centre de Recherche sur les Macromole´cules Ve´ge´tales CERMAV and Joseph Fourier University, BP53, 38041, Grenoble Cedex 9, France Received March 25, 2008; Revised Manuscript Received November 10, 2008
The self-assembly of dextran-block-polystyrene (dex-b-PS) block copolymers was investigated in solution. The hydrophobic PS weight fraction in these block copolymers ranges from 7 to 92% w/w, whereas the average number molar mass of dextran was kept constant at 6600 gmol-1. Self-assembly by direct dissolution in water could be performed only for block copolymers with a low hydrophobic content (7% w/w), whereas mixtures of tetrahydrofuran and dimethylsulfoxide were required for higher PS content, before transferring the structures into water. Core-shell micelles, ovoı¨ds, and vesicles could be identified upon characterization by light and neutrons scattering, atomic force microscopy, and transmission electron microscopy. Most of the morphologies observed were not expected considering the chemical composition of the block copolymers. Finally, the size and shape of these nanoparticles were fixed upon cross-linking the dextran block through reaction of the hydroxyl groups with divinylsulfone. The role of the dextran conformation on the self-assembly process is discussed.
Introduction The self-assembly of block copolymers in a selective solvent provides a great variety of morphologies such as spherical and wormlike micelles, vesicles, and numerous other microstructures. Such a process is the result of two driving forces that interplay oppositely: on the one hand, long-range repulsive interactions between incompatible blocks, and on the other hand, short-range attractive interactions due to the chemical bond linking the two blocks, which leads to microphase rather than macrophase separation. The morphologies observed at thermodynamic equilibrium result from the minimization of the free energy of the self-assembled structures and are controlled by molecular parameters such as the chemical nature of the blocks, the volume fraction of each block, the molar mass and the overall architecture of the copolymer.1 Solution parameters such as polymer concentration, temperature, solvent quality, pH, and ionic strength2 also play a crucial role on the self-assembly. Finally, the dissolution process is another key parameter that impacts morphological aspects of the nanoparticles formed.3-6 The self-assembly phenomenon in solution is therefore extremely complex; numerous theoretical works have been devoted to the subject7-10 as well as experimental studies, which have helped to clarify the micellization process and the way to generate stable morphologies.3,5,11 Discher and Eisenberg11 proposed a general empirical law which stipulates that block copolymers possessing a hydrophilic weight fraction (f) >45% are expected to form spherical * To whom correspondence should be addressed. Tel.: 00 33 5 57 00 36 96. Fax: 00 33 5 57 40 00 84 87. E-mail:
[email protected] (J.F.L.M.);
[email protected] (Y.G.). † Laboratoire de Chimie des Polyme`res Organiques UMR5629. ‡ Centre de Recherche Paul Pascal CNRS-UPR 8641. § Centre de Recherche sur les Macromole´cules Ve´ge´tales CERMAV and Joseph Fourier University.
micelles, while those with f < 25% self-assemble into inverted nanostructures. Block copolymers with hydrophilic weight fraction around 35 ( 10% are predicted to form polymeric vesicles also referred to as polymersomes. Nanostructures formed by self-assembly in solution of block copolymers make them attractive materials in various applications, in particular, as nanocarriers for the encapsulation and controlled release of drugs. For example, polyester-based block copolymers are the subject of numerous studies12-17 aiming at the development of biomaterials or drug delivery systems. In addition to providing abundant sources of raw materials, naturally occurring oligosaccharides and polysaccharides exhibit attractive properties such as biodegradability, biocompatibility, texturing, or gelifying properties. Harnessing these properties in nanostructures obtained from the self-assembly of block copolymers could be of great interest for numerous applications from cosmetics to medicine. Pioneer work in this field has been performed in the early 1980s by Ziegast and Pfannemu¨ller who coupled an oligosaccharide functionalized with an aldonolactone at its reducing end to an R,ω-diamino poly(ethylene oxide) (PEO).18 In recent years, other routes, though scarce, have been proposed to develop new “hybrid” polysaccharide-based block copolymers. A first strategy is based on the coupling of an amino-terminated synthetic polymer to the terminal aldehyde of an oligosaccharide,19-23 possibly followed by the enzymatic growth of the oligosaccharide block.21,24,25 A second route starts from the chemical modification of the reducing ends to subsequently grow the synthetic block by means of a controlled/ living polymerization. In this method, sugar derivatives or cyclodextrine are derivatized into macroinitiators of controlled radical polymerization process26,27 to create “end sugar” functionalized synthetic polymers. Recently, our group has reported a methodology to synthesize dextran-block-polystyrene (dexb-PS) diblock copolymer by atom transfer radical polymerization
10.1021/bm800778n CCC: $40.75 2009 American Chemical Society Published on Web 12/11/2008
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Biomacromolecules, Vol. 10, No. 1, 2009
Table 1. Molecular Characteristics of the Block Copolymers Used in this Study sample
Mn SECa
DPn NMRb
PDIa
ΦPS %w/w
dex40-b-PS5 dex40-b-PS270 dex40-b-PS775
17500 82200 160000
5 270 775
1.4 1.7 1.9
7 81 92
a Determined by SEC in THF (calibration with PS standards). b Overall composition determined by 1H spectroscopy, knowing the molar weight of commercial dextran (Mn ) 6600 g · mol-1).
(ATRP).28 From a synthetic viewpoint, most of reported works take advantage of the terminal aldehyde of the oligosaccharide that is in equilibrium with a hemiacetal function, except in a few references.21,24 However, the number of saccharide based block copolymers systems that have been thoroughly investigated is limited and among existing studies only a few contain informations about their solution behavior. For instance, Liu and Zhang20 have shown by transmission electron microscopy (TEM) and dynamic light scattering (DLS) that dextran-b-poly(ε-caprolactone) self-assembles in water into polydisperse micellar structures with an average diameter of 100 nm. pHresponsive carboxymethyl dextran-b-poly(ethylene oxide) were also shown to self-assemble in aggregates with an average size of 100 nm in acidic media by intra- and intermolecular hydrogen bonding.22 The self-assembly of PEO-b-amylose in chloroform results in the formation of reverse micelles in which the amylose block is believed to adopt a helical structure.29 The behavior of sugar end-functionalized PS (from glucose to maltohexaose) has also been studied in toluene.30 Reverse micelles were obtained with an aggregation number varying with the overall composition of the block copolymer. In a recent addition, the selfassembly of amylose-b-PS in THF revealed that nonequilibrium aggregates with different sizes were obtained,31 whereas crew cut micelles with a diameter ranging from 10 to 30 nm were observed in water. In the latter case, the amylose corona was found to exhibit a rod-like structure. It is important to note that depending on their chemical nature, polysaccharide chains can present a rod conformation (Xanthan, amylose,...) or a coil conformation like, for instance, pullulan or dextran, although the latter one behaves as a rod for molar mass below 2000 g/mol.32 This coil or rod character can have a marked influence on the self-assembly process, as it will be discussed later in the paper. From these findings, it appears difficult to establish general principles with respect to the self-assembly of polysaccharide based block copolymers. For instance, the influence of the helical structure of a polysaccharide such as amylose on the selfassembly process is not yet clearly understood. In the present report, we describe the self-assembly properties of a series of dex-b-PS block copolymers in organic solvent but also in water. The degree of polymerization (DP) of the polysaccharide was kept constant (∼40) but that of PS blocks was varied from 5 to 770. The synthesis of these hybrid block copolymers has been already described in a previous report.28 The influence of the block copolymer composition on the micellar structures formed has been thoroughly investigated by means of dynamic and static light scattering, atomic force microscopy and freeze fracture transmission electronic microscopy. We discuss at the end of this work the possible influence of the dextran block on the self-assembly process.
used were of technical grade. Divinyl sulfone (Aldrich) was used without purification. The following nomenclature was used for the different block copolymers: dexa-b-PSb, where a and b stand for the degree of polymerization of dextran and polystyrene, respectively. Preparation of the Self-Assembled Nanoparticles. The direct dissolution of dex40-b-PS5 in water was achieved after stirring for two days at 90 °C, followed by sonication. The direct dissolution in water for systems with a larger PS content was just not possible. As no common solvent for both PS and dextran could be found, a mixture of THF and DMSO was used to dissolve these diblock copolymers, DMSO being selective for the dextran block and THF for the PS one. This method is slightly different from the procedure generally used that consists in the dissolution of the block copolymer in a common solvent for both blocks, the self-assembly being induced by adding a selective solvent for one of the blocks.2,6,33-35 All dex-b-PS block copolymer samples could be dissolved in DMSO/THF mixtures in a composition range from 50 to 75% in DMSO for dex40-b-PS270 and from 30 to 45% in DMSO for dex40-b-PS775 (dotted lines in Figure 3). In these compositions, no clear scattered signal was detected, indicating that the block copolymers behave as unimers. Then, the solvent composition was changed by addition of either DMSO or THF to induce selfassembly and engineer different morphologies. Cross-Linking Procedure of the Nanoparticles. Divinyl sulfone (DVS) was added under nitrogen to dex40-b-PS270 dissolved in a DMSO/ THF mixture with a composition of 40/60 to cross-link dextran in the core of the nanoparticles or in a DMSO/THF mixture with a composition of 90/10 to cross-link dextran in the shell of the nanoparticles. A large excess (>10 equiv) of DVS was used relative to the hydroxyl groups present in the dextran chains. The reaction was performed at room temperature during 24 h. Atomic Force Microscopy (AFM). Samples for AFM analysis were prepared by evaporation at room temperature on substrates starting from water solutions. Practically, for aqueous media, 20 µL of a dilute solution (∼5 mg/L) was dropped on a 1 × 1 cm2 freshly cleaved mica substrate. Samples were analyzed after complete evaporation of water at room temperature. For solution in THF, 20 µL of a dilute solution (∼0.5 mg/L) was spin-coated on freshly cleaved HOPG. For solution in DMSO, 20 µL (∼5 mg/mL) were dropped on HOPG surfaces and the evaporation of the solvent was performed under dynamic vacuum. AFM images were recorded in air with a dimension microscope (Digital Instruments, Santa Barbara, CA) operated in tapping mode. The probes were commercially available silicon tips with a spring constant of 40 N/m, a resonance frequency lying in the 270-320 kHz range and a radius of curvature in the 10-15 nm range. In this work, both the topography and the phase signal images were recorded with the highest sampling resolution available, that is, 512 × 512 data points. Dynamic and Static Light Scattering. Dynamic and static light 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 and an ALV-5000/EPP multiple τ digital correlator with 125 ns initial sampling time. The copolymer solutions were maintained at a constant temperature of 25.0 ( 0.1 °C in all experiments. All the scattering measurements were carried out from 40 to 120° by steps of 10°. Data were collected using ALV Correlator Control software, and the counting time was fixed at 300 s for each angle. Dynamic light scattering measurements were evaluated by fitting of the measured normalized time autocorrelation function of the scattered light intensity. The data were fitted with the help of the constrained regularization algorithm (CONTIN), which provides the distribution of relaxation times τ, A(τ), as the inverse Laplace transform of g(1)(t) function
g(1)(t) )
Experimental Section Materials. Molecular features of dex-b-PS diblock copolymers previously synthesized28 are reported in Table 1. All organic solvents
33
∫
∞
0
A(τ) exp(-t ⁄ τ)dτ
(1)
Apparent diffusion coefficients D were obtained by plotting the relaxation frequency, Γ (Γ ) τ-1) versus q2, where q is the wavevector defined as
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Biomacromolecules, Vol. 10, No. 1, 2009 q)
θ 4πn sin λ 2
()
Houga et al. (2)
and 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. Single nanoparticle diffusion coefficient were determined by extrapolation to zero concentration and hydrodynamic radius (RH) was calculated from the Stokes-Einstein relation
RH )
kBT 2 kBT q ) 6πηΓ 6πηDreal
(3)
where kB is the Boltzmann constant, Γ is the relaxation frequency, T is the temperature, and η is the viscosity of the medium. The size dispersity of the particles was then calculated by the 〈∆Γ2〉/ Γ2 ratio, in which 〈∆Γ2〉 was determined by analysis of the first-order correlation function by cumulant analysis.
1 1 g1(t)H ) exp[-〈Γ〉t + 〈∆Γ2 〉t2 - 〈∆Γ3 〉t3 + ...] 2 6
(4)
The reduced elastic scattering I(q)/kC, with K ) 4π2n0(dn/dc)2(I090°/ R90°)/λ04NA, was measured in steps from 40 to 120° scattering angle, where n0 is the refractive index of the standard (toluene), I090° and R90° are the intensity and the Rayleigh ratio of the standard at θ ) 90°, respectively, dn/dc is the increment of the refractive index, C is the concentration, and I(q) is the intensity scattered by the sample. Elastic (static) intensity was calculated according to standard procedures using toluene as the standard with known absolute scattering intensity. A curvature of the angular dependence in a Zimm plot is often observed in our self-assembled nanoparticles which present hydrodynamic radii larger than about 100 nm. This curvature becomes largely linearized by the Berry representation.36 The radius of gyration (Rg) and the second virial coefficient (A2) were obtained using the equation
Kc ) R(q, c)
(
1 1 1 + Rg2q2 (1+A2Mwc) Mw 6
)
(5)
(Kc/∆R(θ))1/2 was plotted against (sin 2(θ/2) + kC), with k being an adjustable constant. Extrapolation of the experimental data to zero concentration and zero angle gave the micellar parameters of MW, Rg, and A2. We only considered the calculated radius of gyration in our work. Small Angle Neutrons Scattering (SANS). SANS experiments were performed at the Le´on Brillouin Laboratory (Orphe´e reactor, Saclay) on the PACE spectrometer. Two spectrometer configurations have been used to cover a q range from 5 × 10-3 Å-1 to 0.15 Å-1. The main parameters used to calculate the corresponding resolution functions37,38 are listed in Supporting Information (SI; Table S1). The sample (a solution of dex40-b-PS270 at 10 mg/mL in a mixture DMSO-d/THF-d (40/60)) was introduced into a 5 mm thick rectangular quartz cell. The blank sample was pure DMSO-d/THF-d (40/60) v/v. Data treatment was done with the PAsidur software (LLB). Absolute values of the scattering intensity (I(q) in cm-1) were obtained from the direct determination of the number of neutrons in the incident beam and the detector cell solid angle.39 Contribution due to incoherent scattering of the dex40-b-PS270 solute was determined by plotting q4I(q) versus q4 of the sample signal. At large q values, this plot is linear and its slope gives the incoherent contribution of the solute. The magnitude of this slope was around 0.0065 cm-1 and was subtracted from the data. Freeze-Fracture Transmission Electron Microscopy (FFTEM) Technique. A drop of the water solution of dex-b-PS (0.1 mg/mL) was placed between two copper planchettes of a sandwich holder and freezed by plunging into liquid propane. Sample was then fractured at -150 °C and pressure of the order of 10-6 mBar in a BAF 300 Balzers apparatus. The fractured surfaces were replicated with platinum evaporated at a 45° angle, followed by carbon deposition normal to the fracture surface to increase mechanical strength. The copper planchettes were dissolved in chromerge (a mixture of chromic acid,
Figure 1. Autocorrelation function at 90 ° and time distribution function by CONTIN analysis for dex40-b-PS5 at 0.05 mg/mL in water. Inset: Relaxation frequency vs q2.
sulfuric acid and water). The detached replicas were then rinsed with water and cleaned from copolymer with DMSO/THF mixture, before being collected on the 200 mesh copper grid. Observations by transmission electron microscopy were made with a FEI Tecnai 12 Microscope working at 120 keV. Some nanoparticules were directly observed by TEM after evaporation of the solvent. For that purpose, one drop of solution (∼20 µL) at 0.1 mg/mL was deposited on copper EM grid coated with Formvar film. The solvent was dried at atmospheric pressure at room temperature.
Results and Discussion I. Self-Assembly of a 7% w/w PS Fraction dex-b-PS Copolymer. The solution properties of dex40-b-PS5 with a PS weight fraction of 7% were first investigated. Dilute solutions were analyzed by dynamic light scattering (DLS) at different angles at 25 °C. Figure 1 illustrates the autocorrelation function obtained at a scattering angle of 90° with the corresponding CONTIN analysis. Three main time distributions were observed: the main population exhibits a hydrodynamic radius RH ) 28 nm that was calculated from the diffusion coefficient obtained by plotting the characteristic relaxation frequency versus q2 (inset in Figure 1). The other populations were not observed at all the scattering angles and were thus considered as physically meaningless. Moreover, the few characteristic times that could be extracted were not representative of a diffusive mode, as illustrated in Figure S1, SI. The hydrodynamic radius (28 nm) of the main population is characteristic of micelles formed by self-assembly. These micelles were found to be stable for weeks. Given the block copolymer composition (∼7% w/w PS), core-shell micellar structure with a dextran-based corona oriented toward the continuous aqueous phase is the expected morphology and should be at thermodynamic equilibrium. Indeed, the size and dispersity of these micelles remained unchanged even after prolonged heating time. The formation of spherical objects was confirmed by AFM experiments carried out in a tapping mode (Figure 2): the measured diameter (∼40 nm) being in reasonable agreement with DLS findings. A comparison of the size of the micelles (RH ∼ 28 nm in solution) with the characteristic length of the block copolymer analyzed has been made to check the compatibility with a core-shell structure. Assuming a fully extended conformation and average monomer length of 0.25 nm, the contour length for the PS block would be 1.2 nm. A solvated dextran of the same molar mass than in our block copolymers, presents a
Micelles and Polymersomes
Figure 2. AFM image obtained from a drop of a solution in water at 5 mg/L of dex40-b-PS5 evaporated on a mica surface.
Figure 3. Evolution of hydrodynamic radius vs DMSO volumic fraction for different block-copolymers: (O) dex40-b-PS270, (4) dex40-b-PS775. Lines are guides for the eyes. Dotted curve indicates the area where no scattered signal could be detected. Schematic morphologies are inserted in the graph. Red color illustrates dextran and blue color illustrates polystyrene.
hydrodynamic radius in the range of 2 nm. Therefore, it is expected that the hydrophilic chains in core-shell micelles adopt an extended rather than a coil conformation in water.39 Assuming a fully extended or “rod-like” conformation of dextran and considering a characteristic size of 0.5 nm for each sugar unit, a value of 20 nm (for DP ) 40) for its contour length is obtained. This hypothesis is in good agreement with our experimental observations, confirming the formation of micellar core shell structure, with dextran chains in their extended conformation. II. Self-Assembly of Larger PS Fraction dex-b-PS (>81%) Block Copolymers. In the first instance, each solution was analyzed by DLS at different angles, and the resulting RH were plotted as a function of the solvent composition (Figure 3). As the procedure is based on the addition of THF or DMSO to change the solvent composition, this of course induces dilution of the solution. We have first checked that dilution had no influence on the morphology of the nanostructures. This was realized by diluting up to four times a given solution in which self-assembled structures were obtained, without changing its solvent composition and by checking the invariance of hydrodynamic radius. We can thus conclude that variations observed in Figure 3 are exclusively due to the modification of solvent
Biomacromolecules, Vol. 10, No. 1, 2009
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Figure 4. Autocorrelation function at 90° and time distribution function after CONTIN analysis for a dex40-b-PS270 in THF region (92% THF). Frequency relaxation vs q2 are represented in inset.
mixture. Insets in Figure 3 show a schematic representation of the obtained morphologies in the following three distinct domains: DMSO region, THF region, and water, as discussed in the following sections. In all cases, self-assembly occurred irrespective of the solvent composition, giving self-assembled structures with hydrodynamic radii ranging from 70 to 145 nm. For dex40-b-PS270, RH values of about 140 nm were measured at low DMSO volume fraction. Upon increasing the DMSO content, no scattered signal (dotted curve in Figure 3) could be detected from ∼50 to ∼75% of DMSO. Further addition of DMSO (>80%) brought about the formation of smaller nanoparticles (RH ) 120 nm). For the dex40-b-PS775 sample the behavior was similar with hydrodynamic radii ranging from ∼115 to ∼65 nm, when increasing the DMSO content. The domain with no scattering signal was estimated from ∼30 to 45% DMSO. It has to be noted that the same hydrodynamic radii are obtained, whatever the evolution of this solvent composition (from DMSO to the THF region or from THF to the DMSO region), attesting to the reversibility of the self-assembly process. Given the sizes of the nanoparticles obtained for dex40-bPS775 and dex40-b-PS270 samples and the fact that the RH of dex40b-PS775 is smaller than that of dex40-b-PS270, the formation of spherical core-shell micelles is very unlikely. Thus, further investigations were carried out to identify the type of nanostructures formed in THF and DMSO regions. II-1. BehaVior in THF Region. In a first approach, both DLS and SLS experiments served to assess the Rg/RH value. This ratio is well-known to be indicative of the morphology obtained,36,40 (Rg/RH ∼ 0.77 for a hard sphere, Rg/RH ) 1 for a vesicle, Rg/RH ) 1.7 for a coil), provided the nanoparticles present a relatively narrow size distribution. The different structures were also characterized by AFM and FFTEM. A relatively narrow distribution of relaxation time was detected by DLS in all cases. It is worth mentioning that the slow mode observed in Figure 4 (∼2000 nm) was not reproducible and not representative of a diffusive mode. This can be attributed to the existence of few and nonreproducible aggregates and was therefore considered as physically meaningless. Autocorrelation function and relaxation frequency versus q2 are illustrated in Figure 4 for dex40-b-PS270. Similar results are available as ESI for dex40-b-PS775 (Figure S2, SI). Values of the hydrodynamic radii extracted from these experiments are summarized in Table 2. Static light scattering
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Table 2. Radius or Gyration, Hydrodynamic Radii, and Polydispersity Indexa solvent THF region DMSO region water a
system dex40-b-PS270 dex40-b-PS775 dex40-b-PS270 dex40-b-PS775 dex40-b-PS270 dex40-b-PS775
(92% THF) (95%THF) (95%DMSO) (90%DMSO)
Rg (nm)
RH (nm)
Rg/RH
expected morphology
dispersity index 〈∆Γ2〉/Γ2
200 115 250 77 60 70
145 110 115 77 64 77
1.38 1.04 2.17 1 0.94 0.91
polydisperse vesicles vesicle elongated nanoparticle vesicle vesicle vesicle
0.30 0.20 0.11 0.10 0.074 0.10
Given at 90° (cumulant analysis) obtained by dynamic light scattering.
Figure 6. SANS intensity as a function of q for dex40-b-PS270 in DMSO-d6/THF-d5 (40/60) mixture (10 mg/mL).
Figure 5. (Top) AFM height micrographs obtained in the THF region (92%) for (a) dex40-b-PS270 and (b) dex40-b-PS775 (95% THF). (Bottom) Section profile of (b).
experiments were also performed and a Berry plot was used to extract the Rg values (SI, Figures S4,5). Size dispersity of the objects formed, estimated by the cumulant analysis, is also indicated in Table 2. Considering the light scattering results, dex40-b-PS775 would afford vesicular morphology (Rg/RH ∼ 1), the case of dex40-b-PS270 being less clear. Indeed, the ratio Rg/ RH of 1.4 in that case does not correspond to any known morphology in literature. This can be explained by the relatively high polydispersity (0.30) of the structures obtained. To get more insight into the morphologies obtained, AFM experiments were performed on dex40-b-PS270 and dex40-b-PS775. As illustrated in Figure 5, for both block copolymers, a donuttype morphology is observed. In the case of dex40-b-PS270 (Figure 5a, top), it appears that large number of objects are in the range of ∼100-400 nm in reasonable agreement with the distribution of RH observed in Figure 4. However, it is difficult to evaluate the polydispersity of these objects because of their insufficient number on the picture. In the case of dex40-b-PS775 (Figure 5b, top), less objects are observed, with a characteristic size between 300 nm and 1 µm, in relative good agreement with distribution of RH obtained by DLS (Figure S2, SI). Again, this technique confirms the formation of vesicles (polymersomes) that appear as being adsorbed on the surface. The force induced by the AFM probe causes deformation of the vesicles as verified by analysis of cross section profiles (Figure 5b, bottom). Such a profile was drawn using Monte Carlo simulations on adsorbed lipid vesicles.41 This deformation is presumably due to the presence of residual DMSO, which interacts
with the inner membrane of dextran playing the role of a plasticizer and giving the vesicles the aspect of circular plates on the mica surface. Very similar AFM results have been obtained on vesicles resulting from the self-assembly of discostic liquid crystalline molecules42 and diblock codendrimers based on poly(benzylether) and poly(methallyl dichloride), above the Tg of both blocks.43 In both cases, results were also interpreted in terms of deformation of the spherical vesicle following adsorption on the surface, this deformation being possibly enhanced by the force of the AFM probe. Based on these elements, it can be concluded that dex40-b-PS775 and dex40-bPS270 self-assemble into vesicles when dissolved as unimers in a mixture of DMSO and THF and then further diluted with THF (92% for dex40-b-PS270 and 95% for dex40-b-PS775). It has to be noted that a few very small objects appearing as non hollow structures on AFM images could be due to the existence of spherical micelles which could result from a “residual” kinetic influence on the micellar structure formation. Due to their size and their low number compared to the vesicular morphologies, these objects are not detected in light scattering experiments. The solvent composition (DMSO/THF 40/60) was also investigated in the case of the dex40-b-PS270 sample, to gain more information on the morphology generated before dilution to a high THF content (>92%). The Rg/RH ratio was found roughly the same as in the case of the THF-rich solutions (∼1.4), which is not relevant to draw a clear conclusion about the type of self-assembled nanostructures. SANS experiments were thus performed on a solution that was prepared in this case with a deuterated mixture of solvents (DMSO-d6/THF-d5 40/60 v/v). The corresponding scattering curve is shown in Figure 6. Due to the large diameter and high polydispersity of the particles, the scattered intensity did not present any characteristic oscillation from the form factor that could easily help the data interpretation. However, this curve presented a clear q-2 slope of the scattered intensity as a function of q, attesting to the
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37
Figure 8. TEM image obtained for the dex40-b-PS270 in water (0.1 mg/mL). Figure 7. Autocorrelation function at 90° and time distribution function after CONTIN analysis for dex40-b-PS270 in DMSO region (95% DMSO). Frequency relaxation vs q2 is presented in inset.
existence of a flat interface, which is characteristic of vesicle membranes. We thus conclude that the dex40-b-PS270 copolymer self-assemble in the DMSO/THF 40/60 solution into vesicles with its PS chains oriented toward the solvent. The size and morphology of these vesicles remained unaffected upon addition of THF. II-2. BehaVior in the DMSO Region. Each block copolymer solutions exhibited a transition region where no scattering signal was detected “entering” either the THF- or the DMSO-rich regions. This means that morphologies obtained in THF disassemble into free copolymer chains (unimers) when the solvent mixture solvates efficiently both blocks. These free chains subsequently self-assemble into reverse nanoparticles when DMSO content increases. In this case, the nanoparticles consist of dextran blocks oriented toward the solvent, whereas PS chains are shielded from the continuous DMSO medium in the inner part of the nanoparticles. Each solution of the block copolymers has been studied in the DMSO region by DLS and SLS. Autocorrelation functions and CONTIN analysis are shown in Figure 7 for dex40-b-PS270 as an example, the other results being provided in SI (Figure S3). All the block copolymers exhibit a single relaxation time with a relatively narrow size distribution, as indicated by cumulant analysis (Table 2). The Berry plot are available in SI (Figures S6, S7). One can note that the characteristic sizes of the nanoparticles are lower compared to the ones measured in the THF region. When diluting the THF-rich solution with DMSO, the morphology of the dex40-b-PS270 is modified as the Rg/RH ratio stands above 2, a value that is characteristic of an anisotropic structure. It has to be noted that no rotational diffusion mode in depolarized DLS experiments was detected,44 meaning that the anisotropy of the nanoparticles formed is relatively weak. In the case of dex40-b-PS775, a vesicular structure is expected in DMSO according to its Rg/RH ratio. Unfortunately, no reliable results could be obtained by an imaging technique in the DMSO-rich region to confirm these results. Indeed, evaporating DMSO under vacuum caused the disruption of the nanoparticles formed. In order to further elucidate the exact structure, we then tried to transfer the nanoparticles from DMSO to water. II-3. BehaVior in Water. Starting from solution containing 95% of DMSO and 5% THF, which means that the dextran blocks are oriented toward the solvent and PS chains are shielded
Figure 9. Freeze-fractured TEM image of dex40-b-PS775 (down) and dex40-b-PS270 (up; from water solution, 0.1 mg/mL). Inset: schematic representation of the fracture and platinum shadowing (arrows).
within the core of the nanoparticles, water was added dropwise until a total volume fraction of 50% was reached. The hydrodynamic radii measured under these conditions remained unchanged upon addition of water onto dex40-b-PS775. In contrast, for dex40-b-PS270, the RH value decreased from 115 to 64 nm (Figure 7 and Figure S9, SI). This could be ascribed to a morphology change of the self-assemblies and will be discussed further. Dialysis was performed against distilled water to remove all traces of DMSO and THF. Results of SLS and DLS experiments are given in Table 2, and those drawn from imaging techniques (TEM and FFTEM) are shown in Figures 8 and 9. After dialysis, no significant change of the hydrodynamic radius of the nanoparticles could be noted for both block copolymers. In water, the Rg/RH ratio is close to one, irrespective of the block copolymer composition, suggesting a vesicular morphology. This confirms the occurrence of a morphological change from an anisotropic structure to spherical vesicles for dex40-bPS270 from its initial DMSO-rich solution, where the Rg/RH was higher than 2. Hollow spheres with low dispersity are clearly visible on TEM image (Figure 8), their characteristic sizes being in good agreement with those obtained by DLS. The replica of freeze fractured samples of dex40-b-PS775 and dex40-b-PS270 were further analyzed by TEM (Figure 9). In the case of dex40-bPS775, the holes inside the spheres are clearly evidenced by the presence of a shadow. The fracture seems to occur as schematized in Figure 9. The shadowing by platinum images the vesicular structure of the sample. For the dex40-b-PS270 nano-
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Figure 10. AFM image obtained for cross-linked dex40-b-PS270 nanoparticles from solution DMSO/THF 95/5.
particles, one can notice a black core surrounded by a thick structured ring. We believe that the particles were just cut off in the plane as shown in the inset, which leads the inside of the vesicle to appear black. The images obtained are compatible with the formation of nanoparticles, exhibiting a vesicular structure, and thus confirm the results obtained from scattering experiments. III. Cross-Linking of the Nanoparticles. To better elucidate the morphologies obtained in the DMSO-rich region, we attempted to cross-link them directly from the organic solution. Indeed, cross-linking is an appropriate method to fix the selfassembled morphologies and modify their properties such as their permeability or strength. This can be realized by different approaches that have been recently reviewed,45 with radical cross-linking polymerization and chemical reaction between two functional groups being mainly used. Cross-linking can be performed within the core 46-50 or in the shell of the micellar structures.51-60 If the cross-linking of core shell micelles has been widely investigated, vesicles made from the self-assembly of block copolymer have also been the subject of such treatment. 61-64 In this work, DVS was used to cross-link the dextran chains of the self-assembled nanostructures in DMSO/THF solution. This cross-linker has been successfully used in polyacrylate derivatives54,65 or to cross-link vesicles made from the self-assembly of amphiphilic comb-like PEG.66 In DMSO/ THF medium, nanoparticles with a core constituted of dextran or PS can be obtained reversibly, depending on the composition of the solvent mixture. The cross-linking of dextran was successfully accomplished irrespective of the location of dextran in the nanoparticle (in the inner core or as the shell). To this end, a solution of dex40-b-PS270 in DMSO/THF (40/60) was prepared to obtain a vesicle with dextran oriented in the inner membrane before adding DVS. The evolution of the nanoparticles was then followed by DLS. A slight increase of RH (from ∼140 to ∼155 nm) was observed, which could be explained by an increase of the rigidity of the dextran membrane, resulting in a slight decrease of its curvature. Dilution with THF of this solution left RH almost unchanged, confirming the stability of the nanoparticles with their PS chains located at the surface of the vesicle. In contrast, a progressive addition of DMSO induced the irreversible precipitation of the cross-linked nanoparticles. Indeed, the structure cannot disassemble and form stable reverse vesicles with dextran oriented toward the continuous medium because of intermolecular cross-linking of dextran. The same approach was used for nanoparticles presenting dextran at the surface. A solution of dex40-b-PS270 was prepared in 90/10 DMSO/THF, thereafter, DVS was added. A marked increase of the hydrodynamic radius from 115 to 140 nm is again observed, and the subsequent dilution of this solution with THF brought about the particles precipitation, attesting to the efficiency of the cross-linking process.
Figure 11. AFM image obtained for cross-linked dex40-b-PS270 nanoparticles after dialysis in water.
In the case of dex40-b-PS270, we anticipated the formation of an anisotropic (elongated) self-assembled structure in DMSO. This hypothesis could not be confirmed by imaging techniques. Indeed, as indicated above, the evaporation of DMSO under vacuum led to a disruption of the nanoparticles. After crosslinking, this could be done without destroying the nanoparticles. AFM imaging was then performed as shown in Figure 10. Ovoïd nanoparticles are clearly observed with characteristic dimensions of ∼250 nm along the main axis and ∼100 nm perpendicular to it, confirming the anisotropy and the size measured by scattering techniques. Detailed information about these anisotropic objects, whether they are filled or hollow structures, have not yet been obtained. However, as vesicles were formed from noncross-linked samples upon addition of water and dialysis (see Figure 9 and Table 2), the formation of hollow anisotropic objects can be contemplated here. Upon addition of water (up to 50% v/v) and dialysis, these cross-linked anisotropic nanoparticles became spherical as shown in Figure 11. The characteristic size is in reasonable agreement with the one obtained by dynamic and static light scattering on the cross-linked system in water (RH ) 145 nm; Rg ) 210 nm; Rg/RH ) 1.44, see Figure S11). The Rg/RH ratio is different from what is expected for a vesicular structure. At this point, it is difficult to conclude on the morphology obtained in water from cross-linked samples. We think that vesicular structure with relatively pronounced size dispersity could be possible. Finally, these results suggest that in spite of cross-linking, the mobility of some dextran chains increases when DMSO is replaced by water, the constraints and the stresses existing in the anisotropic structure being likely relaxed by a morphology change. For noncross-linked morphologies, the phenomenon is supposed to be the same but with
Micelles and Polymersomes
greater amplitude, as a decrease of hydrodynamic radius from 115 to 65 nm is observed when water is added to a solution of dex40-b-PS270 in DMSO. Obviously, it is difficult to describe precisely what really happens at the molecular level during the transfer from DMSO to water as the morphology in DMSO is not yet fully elucidated (full or hollow ovoïds).
Conclusions The self-assembly process of dex-b-PS diblock copolymers in organic and aqueous media is strongly influenced by their overall composition. The reversibility of the self-assembly process (from DMSO to THF and vice versa) demonstrates that some thermodynamic equilibrium can be reached. Core-shell micelles are generated in water for a content of dextran equal to 93% (w/w). In all other conditions, block copolymers selfassemble into vesicular structures. These morphologies were not expected and do not correspond to the prediction and classification proposed by Discher and Eisenberg11 (e.g., for hydrophilic fraction of 19 and 8%) for coil-coil type block copolymers, because crew-cut micelles would have been expected. Although dextran is considered to exhibit a coil conformation, it can adopt a rod-like conformation for molar masses below 2000 g/mol.32 Because the average molar mass of the dextran in this study is equal to 6600 g/mol and its polydispersity index is around 1.6, it is obvious that some of the dextran chains are below the 2000 g/mol range and accordingly adopt a rod conformation. It is well-known in the self-assembly of rod-coil block copolymer that their stiffness asymmetry promotes self-assembly into flat membranes and in diluted state, into vesicles.67-70 Therefore, the slight stiffness asymmetry induced by the dextran chains that adopt a rod conformation could play a crucial role in the formation of a planar bilayer membrane, resulting in the formation of vesicles either in water, THF, or DMSO. The study of block copolymers with longer dextran chains and narrow size distribution could bring interesting information about this aspect. This study revealed some specificities of dextran-based block copolymers (unexpected vesicles, ovoïds,...) and that further studies are needed to get a better insight, especially on the influence of the dextran conformation to the self-assembly behavior. Indeed, if vesicular structure could be explained by a rod conformation of short dextran chains, the origin of the formation of the ovoı¨d structures is not yet understood. In addition, the exact role of the dextran chains in the morphological transition observed during transfer from DMSO to water has to be further elucidated. Morphologies obtained in DMSO could be transferred in water paving the way of potential applications as carriers or vehicles. The vesicles (or polymersomes) obtained are particularly interesting because of their ability to encapsulate both hydrophobic and hydrophilic molecules. The morphologies can be efficiently cross-linked via the hydroxyl groups of the dextran, which can be a way to tune the membrane permeability of these vesicles. Moreover, low concentration regime can be reached without disruption of the nanoparticles, which could be an advantage in many applications. Acknowledgment. The authors would like to thank Prof. Olivier Mondain Monval (CRPP, Bordeaux) for helpful comments on freeze-fractured TEM images, Michel Schappacher for AFM measurements, Annie Bruˆlet (LLB, CEA Saclay) for SANS measurements, and referees for their helpful comments. Supporting Information Available. Experimental details about light scattering measurements, experimental details about
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the transfer of the morphologies from organic solvent to water, as well as dynamic and static light scattering results. This material is available free of charge via the Internet at http:// pubs.acs.org.
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