Hierarchical Structuring of Liquid Crystal Polymer–Laponite Hybrid

Article pubs.acs.org/Langmuir

Hierarchical Structuring of Liquid Crystal Polymer−Laponite Hybrid Materials Ulrich Tritschler,† Igor Zlotnikov,‡ Paul Zaslansky,∥ Barbara Aichmayer,‡ Peter Fratzl,‡ Helmut Schlaad,*,§ and Helmut Cölfen*,† †

Physical Chemistry, University of Konstanz, Universitätsstraße 10, D-78457 Konstanz, Germany Department of Biomaterials and §Colloid Chemistry, Max Planck Institute of Colloids and Interfaces, Research Campus Golm, D-14424 Potsdam, Germany ∥ Berlin-Brandenburg Center for Regenerative Therapies/Julius Wolff Institute, Charité - Universitätsmedizin Berlin, D-13353 Berlin, Germany ‡

S Supporting Information *

ABSTRACT: Biomimetic organic−inorganic composite materials were fabricated via one-step self-organization on three hierarchical levels. The organic component was a polyoxazoline with pendent cholesteryl and carboxyl (N-Boc-protected amino acid) side chains that was able to form a chiral nematic lyotropic phase and bind to positively charged inorganic faces of Laponite. The Laponite particles formed a mesocrystalline arrangement within the liquidcrystal (LC) polymer phase upon shearing a viscous dispersion of Laponite nanoparticles and LC polymer in DMF. Complementary analytical and mechanical characterization techniques (AUC, POM, TEM, SEM, SAXS, μCT, and nanoindentation) covering the millimeter, micrometer, and nanometer length scales reveal the hierarchical structures and properties of the composite materials consisting of different ratios of Laponite nanoparticles and liquid-crystalline polymer.

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INTRODUCTION In natural organic−inorganic biomineral composites such as bone and nacre, stiff but very brittle mineral crystals are joined by soft and ductile organic materials. 1,2 Sophisticated hierarchical structuring and well-controlled coupling of the interface between the two components are key elements leading to a combination of stiffness and toughness of the material,3,4 thus solving the conflict between strength and toughness.5 A staggered array of anisotropic particles (plates, fibers, etc.) enables an optimum combination of stiffness and toughness.4,6 The stiffness of the composite materials arises from a high fraction of stiff particles, whereas deformation is mainly based on the shearing of the organic component in which the stiff particles are embedded.6,7 The most studied system of this kind is nacre, which is 3000 times more fractureresistant than the aragonite CaCO3 polymorph that constitutes 95% of this material.2 The influence of hierarchical structuring of biomaterials on their outstanding mechanical performance was recently outlined by Meyers et al.8 Inspired by the structure and outstanding properties of biominerals, many research groups have undertaken the major challenge of developing biomimetic lamellar composite structures. One of these interesting topics is the development of artificial nacre, the structure and mechanical properties of which can be mimicked by using clay minerals, montmorillonite, and Laponite clay platelets. © XXXX American Chemical Society

Clay minerals are layered silicates often appearing as anisotropic plate-shaped nanoparticles that can act as a mesogen. Consequently, clay minerals were found to form lyotropic colloidal liquid crystals (LC).9,10 Investigations of aqueous suspensions of Laponite nanoplatelets, a synthetic clay with positively charged rims and negatively charged exposed faces,11 revealed the formation of a nematic LC phase,12 which was demonstrated by small-angle X-ray (SAXS) studies.13 Large-scale ordering of an aqueous Laponite suspension near the air−Laponite suspension interface was reported by Joshi and co-workers.14 The behavior of suspensions of Laponite mixed with mesogenic hosts such as thermotropic liquid crystal K15, showing large-scale structures of aggregates, was investigated. The aggregates consist of mixtures of single and stacked self-organized platelets15 or cholesteric liquid crystals, revealing a stabilizing effect on cholesteric planar textures.16 Because of this self-organization into arrays of parallel-ordered clay platelets and the morphological similarity to the micrometer-sized aragonite platelets in nacre, bioinspired nacre composites were fabricated by using nanoplatelets, in particular, clays. The advantage of the nanosized clay particles compared Received: March 8, 2013 Revised: May 10, 2013

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self-organization and self-alignment of LC polymers were developed to mimick the optical and structural properties of golden beetle Chrysina resplendens.42 Inorganic materials with defined porous structuring were obtained by means of selfassembled lyotropic liquid crystals.43−47 Materials combining mesoporosity and chiral organization formed by using chiral surfactants48,49 or by using nanocrystalline cellulose,50 resulting in chiral long-range orientation, were obtained. We propose a model system for organic−inorganic composite materials synthesized via one-step self-organization on three hierarchical levels. Statistical copolymers carrying cholesteryl side chains form two levels of hierarchical structuring through the formation of a chiral nematic LC phase. The polymer also carries carboxyl chains (N-Bocprotected amino acids), which act as gluing units to bind to the Laponite nanoplatelets with positively charged rims. The Laponite nanoplatelets, consisting of the inorganic phase, build up the third level of hierarchical structuring via mesocrystal formation.

to the microsized aragonite platelets in nacre is that materials become insensitive to flaws on the nanoscale.17 Bonnet et al. reported the preparation of artificial nacre via the self-assembly of Na/Ca montmorillonite platelets, which was obtained by the evaporation of dilute aqueous dispersions of delaminated platelets.18 Another approach to mimicking nacre was the layering of montmorillonite clay platelets and polyelectroyles, as described by Ozturk et al.19 and Kotov et al.,20 silica and polyelectrolytes, as described by Char et al.,21 and CaCO3 and polymers22,23 via layer-by-layer assembly, finally leading to clay−polyelectrolyte multilayers. Investigations of the influence of the size and surface area of different clays are reported by Stefanescu and Negulescu et al.,24 who prepared multilayered films consisting of poly(ethylene oxide) (PEO) and montmorillonite clay platelets or PEO and Laponite clay platelets. Polymer−clay nanocomposites were also synthesized by mixing poly(vinyl alcohol) (PVA) and montmorillonite platelets and subsequent doctor-blading.25 Similar paper-making technology could be used to produce multilayered clay−polyelectrolyte papers with additional fireretardant properties26 besides advantageous mechanical properties. Shigehara et al.27 observed the formation of a disconematic liquid-crystalline structure of Laponite nanoparticles within an amorphous PEG matrix in nanocomposites obtained by solution casting. Bonderer et al.28 reported the preparation of multilayered hybrid films combining both high tensile strength29 and ductile behavior29 via a bottom-up colloidal assembly of alumina platelets within a chitosan matrix. Ceramic sintering30 and ice-templating31 approaches were also used to produce layered nacre mimic materials. Nacre mimic materials with a fracture toughness outperforming that of natural nacre and the highest fracture toughness reported to date were reported by Munch et al.30 Another approach using microelectromechanical systems technology was applied to produce microcomposites consisting of silicon particles and a polymeric photoresist, mimicking the crossed-lamellar microstructure of mollusc shells. This shows significant strength and work of fracture as well as some energy-dissipating cracking patterns such as bridged cracks.32 Very recently, Erb et al.33 succeeded in synthesizing hierarchically reinforced, wear-resistant materials by using reinforcing particles of micrometer size coated with superparamagnetic nanoparticles, thus controlling the orientation and distribution of the reinforcing particles by weak magnetic fields. In a similar approach, when composites from polyurethane-based thermoplastic polymers, Laponite nanoplatelets, and alumina microplatelets were synthesized, materials with remarkable mechanical properties were obtained.34 In this paper we present an alternative 'one pot' scalable synthesis approach, where we create hierarchically organized composite structures inspired by findings in natural materials. Hierarchical structuring of polymers was achieved by the bottom-up synthesis of hierarchical structures via the selfassembly of peptide-based block copolymers35 or rod−coil block copolymers36 from solution. A structuring of nanoparticles was obtained by self-assembled hierarchical block copolymer phases.37 The organization of anistropic nanoparticles forming a liquid-crystalline phase can be almost perfect, resulting in mesocrystals that are nanocrystal superstructures with 3D mutual order in the building units.38,39 One such example was reported for V2O5.40 The concept of self-assembly via liquid-crystal formation is found in many biological systems.41 Recently, arrangements of

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RESULTS AND DISCUSSION Polymer Synthesis and Modification. Poly[2-(3-butenyl)-2-oxazoline] (PBOx) was prepared by the cationic ringopening polymerization of 2-(3-butenyl)-2-oxazoline, initiated by methyl triflate, in acetonitrile solution at 70 °C; the reaction was quenched after 3 days with NaOH.51 According to MALDI-ToF MS and SEC analyses (data not shown), isolated PBOx exhibited a number-average molecular weight of Mn ≈ 15 kg mol−1 (average number of repeat units, n ≈ 120) and a dispersity of 1.2. The PBOx was then modified with thiocholesterol (CholSH) (LC unit) and Boc-L-cysteine (Boc-Cys) (“gluing” unit) by applying thiol−ene photochemistry.51 A mixture of PBOx, Chol-SH, and Boc-Cys ([CC]0/[Chol-SH]0/[Boc-Cys]0 = 0.4:0.3:0.3) in THF was irradiated with UV light (λ > 300 nm) at room temperature for 24 h. The product was purified by dialysis against methanol (to remove unreacted thiol) and isolated by freeze-drying from benzene. The composition of isolated copolymer P1 (general chemical structure is given in Scheme 1) was found to be [CC]/ Scheme 1. Simultaneous Modification of Poly[2-(3-butenyl)2-oxazoline] with Thiocholesterol and Boc-L-cysteine

[Chol]/[Cys] = a/b/c = 0.73/0.09/0.18 by 1H NMR spectroscopy (considering the integrals of characteristic proton signals at δ 5.8 (−CH), 5.3 (−CH Chol), and 5.6 (NH Cys), cf. Figure 1). The conversion of double bonds was far less than expected (27% instead of 60% upon quantitative addition of thiols), and the copolymer contained 2 times more Cys units than Chol units (2:1 instead of a 1:1 ratio). The thiol−ene modification of PBOx was repeated in the presence of a photoinitiator, 2,2-dimethoxy-2-phenyl acetophenone (DMPA), [DMPA]0/[CC]0 = 0.5 or 1%, to yield samples P2 and P3, respectively. The compositions of these two copolymers were found to be a/b/c = 0.50/0.21/0.29 (P2) and B

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quantitative assignment is possible for a constant thickness of the polymer film, which can be estimated for a polymer film placed between two glass plates at least to submillimeter precision (checked by means of a profilometer, data not shown). Figure 3 shows the Abrio images of the LC phase of P4

Figure 1. 1H NMR spectrum (400 MHz, CDCl3) of LC “gluing” copolymer P4.

Figure 3. Quantitative birefringence optical micrograph (Abrio) image of shear-induced lyotropic phase formation of LC gluing copolymer P4. (a) 50+ wt % in CHCl3; (b, c) 50+ wt % in DMF.

0.52/0.21/0.27 (P3) (1H NMR). Evidently, with the aid of DMPA, the addition of Boc-Cys to the double bonds came almost to completion (90−97%) and that of Chol-SH reached 0.21/0.3 = 70%. Copolymers P2 and P3 were combined in one sample, P4 (= 0.65 g P2 + 0.60 g P3, a/b/c = 0.51/0.21/0.28); the 1H NMR spectrum of P4 is shown in Figure 1. Lyotropic Phase Behavior. The behavior of LC gluing copolymer P4 was investigated by polarized optical microscoy (POM). A small amount of dry polymer was placed between two glass slides, and then a few drops of CHCl3 were allowed to diffuse into the sample. POM analysis (Figure 2) reveals the

in CHCl3 (a) and in N,N-dimethylformamide (DMF) (b, c) after shearing. It reveals domains with the same color, and thus the same structural orientation, on the length scale of several hundreds of micrometers. Characteristics of the LC phase of P4 on the nanometer length scale were analyzed by small-angle X-ray scattering (SAXS). A 30 wt % solution of P4 in DMF was laterally sheared between two glass slides in one direction. After complete drying of the bulk samples, SAXS measurements of the polymeric liquid-crystalline phases revealed distances of approximately 6, 3, and 1.5 nm attributable to polymer interchain distances and distances between the side chains. Laponite−Polymer Composites. Two dispersions containing Laponite/P4 (1:1 and 2:1 w/w) in DMF/water 1:2.56 v/v were prepared as described in the Experimental Section. The transparent dispersions were stirred overnight, enabling the polymer to bind to the Laponite nanoparticles. A 3 wt % aqueous dispersion of Laponite exhibits a pH of ∼8 to 9. At this pH, the carboxyl groups of P4 are expected to be negatively charged. Consequently, the carboxyl groups are able to bind to the positively charged edges of the Laponite nanoplateles, forming an organic−inorganic hybrid material that was exfoliated in DMF by using a solvent-exchange procedure described by Liff et al.54 Binding between Laponite and polymer in DMF was demonstrated by Analytical Ultracentrifugation (AUC) sedimentation velocity experiments using interference optics. Obtained quantities are the sedimentation coefficients, s (in units of S (Svedberg) = 10−13 s (seconds)), and apparent sedimentation coefficient distributions, ls-g*(s). The sedimentation coefficient is proportional to the size and density of a particle, and ls-g*(s) is the non-diffusion-corrected distribution of sedimenting species, which is an apparent measure of the concentration of the respective component. Results for Laponite/P4 (1:1 and 2:1 w/ w) and reference samples Laponite and P4 in DMF are shown in Figure 4. For these experiments, P4, Laponite, and Laponite/ P4 mixtures (1:1 and 2:1 w/w) were prepared at higher dilution with a final polymer concentration of 1 mg mL−1 in DMF. Compared to the peak of Laponite nanoparticles at s ≃ 47 S, the peaks of the composite materials are shifted toward higher s values to either ∼61 S (1:1 w/w) or ∼70 S (2:1 w/w) (at 20K rpm, Figure 4a). The shift toward higher s values indicates the existence of Laponite agglomerates formed upon interaction and bridging between Laponite particles and

Figure 2. Penetration scan of LC gluing copolymer P4 acquired by POM at 50× magnification.

formation of a chiral nematic lyotropic phase indicated by the double-spiraled texture characteristic.52,53 The distance of ∼0.8 μm between striae corresponds to the half helical pitch of the chiral nematic lyotropic phase. Upon lateral shearing of the specimen in one direction, the cholesteryl-functionalized side chains of P4 in CHCl3 align to form lyotropic phases on longer length scales. In contrast to conventional POM, the quantitative birefringence imaging microscopy technique (Abrio) enables us to quantitatively study the orientation of polymeric LC patterns. Quantitative information about every image point was obtained by measuring the magnitude of retardance and the azimuthal data at every pixel in the charge-coupled-device image. Different structural orientations are depicted by means of different colors, whereas the color saturation corresponds to the degree of orientation as shown by the color wheel. This C

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Figure 4. AUC sedimentation coefficient distributions ls-g*(s) of P4 (black), Laponite (red), Laponite/P4 1:1 w/w (green), and 2:1 w/w (blue) as obtained from sedimentation velocity experiments at (a) 20K rpm and (b) 60K rpm at 25 °C. Figure 5. Quantitative birefringence optical micrographs (Abrio). Laponite/P4 composite materials with Laponite/P4 = 1:1 w/w (upper row) and 2:1 (lower row). Composites were sheared by rotation (left column) or by lateral shearing (right column).

polymer chains. Consistent with this assumption, the shift in s values increases with the increasing fraction of Laponite. The higher the concentration of Laponite, the more nanoparticles bind to the polymer chains and the larger the agglomerates. By scaling the intensity of the P4 reference sample corresponding to a polymer concentration of 1 mg mL−1 (s ≈ 1.5 S at 60K rpm, Figure 4b) and by the integration of multipeak Gauss fits, we found that the fraction of non-bound polymer decreases with increasing amount of Laponite (∼28% and ∼4% free polymer chains for Laponite/P4 1:1 and 2:1 w/w, respectively). The peaks at s ≈ 4.6 S (1:1 w/w) or s ≈ 5.8 S (2:1 w/w) could not be assigned but may be due to a species resulting from the binding of polymer chains to impurities in commercial Laponite. Hierarchically Structured Composite Materials. For the preparation of hierarchically structured organic−inorganic composite materials, dispersions in DMF consisting of Laponite/P4 1:1 w/w and 2:1 w/w were sheared by either lateral shearing between two glass slides in one direction or by slow and controlled rotation by means of a motor using a defined distance. The transparent Laponite/P4 composite materials were analyzed on the micro- to millimeter scale by POM and a quantitative birefringence imaging microscopy (Abrio), on the micrometer scale by X-ray microtomography, and on the micro- to nanometer scale by scanning electron microscopy (SEM), SAXS, and transmission electron microscopy (TEM). As for the polymeric lyotropic phases, the Abrio images of the Laponite/P4 composite materials, reveal the presence of lyotropic regions with the same colors indicating the same structural orientations on the length scale of several hundred micrometers (the prerequisite of constant film thickness on the submillimeter scale was checked by means of a profilometer, data not shown), independent of the ratio of organics to inorganics and independent of the shearing method as well as shearing forces applied (manual lateral shearing or rotational shearing by means of a shear cell; see the Experimental Section for more details) (Figure 5). These regions are considerably larger than the ones found just for the polymer alone (Figure 3). A phase-contrast-enhanced monochromatic (10 KeV) radiograph and a slice in a tomographic reconstruction of a Laponite/P4 1:1 w/w composite, obtained after shearing by rotation, is shown in Figure 6. The radiograph (Figure 6a) shows a projection through the sample, where the phasecontrast enhancement is seen mainly at the edges, suggesting

Figure 6. Phase-contrast-enhanced monochromatic (10 keV) radiograph (a) (gray-level intensities correspond to the extent of transmission where 1.00 corresponds to complete transmission and values higher than 1.00 are due to interference fringes localizing at interfaces) and a tomographic reconstruction slice (b) of a composite sample consisting of Laponite/P4 1:1 w/w obtained after shearing by rotation.

that no significant variations in density exist internally, as already indicated by SAXS analysis using a beam size of 400 μm. A typical cross-sectional virtual slice through the reconstructed volume is shown in Figure 6b, where the rotationally sheared Laponite/P4 1:1 w/w composite surface is on the top and the sample was fixed below. A movie rendering of this data (Supporting Information) shows top and side views of the sample and reveals the topography and irregular surface texture. SEM analysis of the Laponite/P4 composites (images of the 2:1 w/w sample shown in Figure 7) indicates that specimens are composed of layers possessing a thickness of only ∼50 nm, which is below the resolution revealed by microtomography. SAXS measurements were performed to obtain structural information from the specimens with an incident X-ray beam perpendicular and parallel to the expected orientation of Laponite platelets (shearing direction). Figure 8 presents representative 2D SAXS patterns from a Laponite/P4 1:1 w/ w sample produced by rotation. With the beam perpendicular to the rotational direction, an isotropic pattern was obtained (Figure 8a), suggesting that the Laponite platelets are facing the beam. With the beam parallel to the rotational direction, the 2D D

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therefore in the direction perpendicular to the Laponite platelet surface. To a first approximation, the radially averaged SAXS intensity of a stack of cardlike Laponite platelets can be expressed as I(Q) = (2π/Q2)I1(Q), where I1(Q) is the squared Fourier transform of the electron density variation in the direction perpendicular to the platelets.55 We therefore included a Kratky plot, Q2I(Q), in the inset of Figure 9. This graph shows clearly visible maxima that can be directly interpreted as 2π/d, where d is the mean spacing between stacked Laponite platelets. For pure Laponite, we find a d spacing of ∼1.3 nm. With the addition of polymer, this peak shifts to smaller Q values corresponding to d spacings of ∼1.5 nm (Laponite/P4 2:1 w/w) and ∼1.8 nm (1:1 w/w). When comparing integrated intensities for the sample Laponite/P4 1:1 w/w of SAXS data collected with the beam perpendicular and parallel to the shearing direction (dashed blue line and solid blue line in Figure 9, respectively), a shoulder at Q of approximetely 0.25 nm−1 appears. This value corresponds to a d spacing in the size range of the known diameter of the platelets (∼25 nm) suggesting edge-to-edge packing. At the same time, the shoulder corresponding to the stacking of the platelets (d) dissapears. Therefore, we conclude that the Laponite platelets form a closely packed columnar LC phase that is embedded in a polymer matrix (sketch in Figure 9). In every column, the thickness d of the polymeric layer between the platelets depends on the polymer to Laponite ratio. Indeed, the greater the amount of polymer used, the larger the thickness of the polymeric layer between Laponite platelets. Additionally, the shear-induced hierarchically organized structuring of this composite material was visualized via TEM (Figure 10).

Figure 7. SEM images of the Laponite/P4 2:1 w/w composite obtained after shearing by rotation. Images of the cross section (breaking edge) of the composite reveal the layer structure on the length scale of ∼50 nm, which is illustrated by increasing the magnification from left to right.

Figure 8. Representative SAXS 2D patterns from Laponite/P4 1:1 w/ w composites prepared by rotation, obtained with the incident beam perpendicular (a) and parallel (b) to the shearing direction.

pattern is anisotropic (Figure 8b), indicating the orientation of the platelets as portrayed in Figure 8c. Similarly, anisotropic patterns were observed for all specimen preparations regardless of the Laponite content. The SAXS intensity from Figure 8 was radially integrated to obtain information on the packing of the Laponite platelets. Parallel to the shearing direction (solid lines in Figure 9), a shoulder is visible in the region of Q = 3−5 nm−1, which cannot be explained by the form factor of the Laponite platelets computed on the basis of their known shape. From the Q region where the shoulder appears, we found that the stacking distance between platelets must be on the order of 1 nm and

Figure 10. TEM image and corresponding electron diffraction (ED pattern assigned according to Neumann et al.56) of a cross section of Laponite/P4 2:1 w/w prepared via shearing by rotation. The inset of the TEM image shows columnar structuring of Laponite nanoparticles as observed via SAXS (inset scheme in Figure 9).

The electron diffraction (ED) of a cross section of the Laponite−polymer composite (shown for Laponite/P4 2:1 w/ w in Figure 10) with a thickness below 100 nm confirms the superstructure observed by SAXS analysis with a high mutual orientation of the Laponite nanoplatelets along the 130/200 axis (inset scheme of the columnar structuring of the Laponite platelets in Figure 9).56 The spots of ED in the image on the right are slightly deformed into arcs, indicating a slight misaligment (angle of ∼13°) of the superstructure. However, we cannot exclude the possibility that the misorientation observed is an artifact due to mechanical stress induced during TEM sample preparation. By combining the characteristics of

Figure 9. Representative SAXS plots of Laponite reference and Laponite/P4 1:1 w/w and 2:1 w/w composites prepared by rotation. Solid and dashed lines represent integrated data obtained parallel and perpendicular to the rotational direction, respectively. (Inset) Kratky SAXS plot of the same data. E

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formation of homogeneous material on the micrometer length scale. Analysis by SEM, SAXS, and TEM revealed homogeneous structuring on the nanometer length scale. The Laponite nanoplatelets were found to be arranged in columns and to possess a layered structure, finally forming mesocrystalline arrangements. The larger the amount of polymer used, the more polymer chains are able to glue to the edges and thus the larger the observed distance is between the nanoplatelets. The approach is transferrable to other anisotropic particles such as vanadia and gold rods (unpublished work).

the samples observed by SAXS and TEM with the layered structuring observed by SEM, we conclude that a mesocrystalline arrangement of Laponite nanoparticles was formed.38,39 μCT and POM indicate homogeneous distributions of nanoparticles and polymer as well as long-range orientation over several hundreds of micrometers observed on the micrometer length scale. In addition to structural characterization, mechanical characterization using nanoindentation confirmed the existence of anisotropy. The values of reduced elastic modulus and hardness (∼8.5 GPa and ∼0.45 GPa, respectively) measured in the direction parallel to the columns were almost identical for both Laponite to polymer ratios. The properties increased when measuring in the direction perpendicular to the columns, to a reduced modulus and hardness of ∼11 and ∼0.5 GPa in the case of Laponite/P4 1:1 w/w and ∼14.5 and ∼0.6 GPa in the case of Laponite/P4 2:1 w/w, respectively. The obtained results are comparable to previously reported nanoindentation measurements on oriented polymer/Laponite composites prepared by the layer-by-layer technique. For example, Patro et al.57 reported for PVA/Laponite composites a reduced modulus and hardness of 10.3 and 0.48 GPa, respectively. Vertlib et al.58 reported for PDDA/Laponite a reduced modulus and hardness of 6.7 and 0.38 GPa, respectively. Note that in both cases nanoindentation was performed only in one direction, perpendicular to the surface of the platelets.

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EXPERIMENTAL SECTION

Chemicals and Materials. Chemicals and solvents were purchased from several suppliers and used as received (unless otherwise noted). 2-Chloroethylamine hydrochloride (99%), Nhydroxysuccinimide (98%), methyl triflate (99%), thiocholesterol (Chol-SH), and 2,2-dimethoxy-2-phenyl acetophenone (DMPA) were purchased from Aldrich. 4-Pentenoic acid (98%) was purchased from Alfa Aesar. 1-(3-Dimethylpropyl)-3-ethylcarbodiimide HCl (EDAC, 99.4%) was purchased from Iris-Biotech. N-(tert-Butyloxycarbonyl)-Lcysteine (Boc-Cys) was purchased from Merck. 2-(3-Butenyl)-2oxazoline was synthesized in three steps starting from 4-pentenoic acid according to Gress et al.51 Laponite RD was kindly donated by Rockwood Clay Additives GmbH, France. Poly[2-(3-butenyl)-2-oxazoline]. Methyl triflate (40 μL, 0.37 mmol) was added to a solution of 2-(3-butenyl)-2-oxazoline (4.5 g, 36 mmol) in 14 mL of acetonitrile (freshly distilled from CaH2) under an argon atmosphere; the mixture was stirred for 3 days at 70 °C. Then, 1 M aqueous NaOH (1.1 mL) was added, and the mixture was stirred for another 5 h at 70 °C. The polymer was obtained after dialysis against water (MWCO 1 kDa) and freeze-drying. Yield: 3.8 g (84%). Polymer Modification (Example Procedure). A mixture of poly[2(3-butenyl)-2-oxazoline] (0.50 g, 4 mmol CC), Chol-SH (0.48 g, 1.2 mmol), and Boc-Cys (0.27 g, 1.2 mmol) in tetrahydrofuran (THF, 10 mL) was irradiated with UV light (mercury medium pressure UV lamp, Heraeus TQ 150, glass filter) for 24 h at room temperature. The reaction mixture was dialyzed against methanol and evaporated to dryness, and the residual solid was freeze-dried from benzene. Yield 0.7 g. Polymer/Laponite Composites. Laponite was dispersed in Milli-Q water (3 wt %, pH ∼8 to 9) by ultrasonication for 30 min and subsequent stirring for 8 h. After the addition of LC-gluing polymer P4 in DMF (Laponite/P4 = 1:1 or 2:1 w/w; water/DMF = 2.56:1 v/v), the reaction mixture was stirred vigorously overnight, allowing the nanoparticles to bind to the polymer. Water was then removed by evaporation in a rotavapor at 40 °C and 40 mbar to yield a transparent viscous fluid. Gravimetric analysis was used to ensure that only negligible amounts of water remained in the final suspension. Sample Preparation. Laponite/P4/DMF was placed on alumina foil and sheared by either lateral shearing between two glass slides in one direction manually or by rotational shearing by means of a shear cell. The shear cell consisting of a motor-driven disk (Getriebemotor 3000:1 4.5−15 V 540er driven motor purchased from Conrad) and a distance holder enables constant rotational shearing forces by applying a constant rotational speed of 680° per minute clockwise and by using a distance holder of 0.4, 0.5, or 1.5 mm (depending on the desired final size of the composite). After the motor-driven disk was pressed against the distance holder with the viscous hybrid material in its center by the exertion of normal forces in the range of ca. 1−3 N and after the material was sheared, the disk was lifted, allowing the solvent to evaporate. Binding between Laponite nanoparticles and P4 was investigated via AUC sedimentation velocity experiments. Syntheses of Laponite/LC copolymer hybrid materials for these experiments were performed analgously, however, starting from Laponite dispersions of 1 mg mL−1 and finally by diluting the hybrid dispersions to 1 mg mL−1, with respect to the polymer, in DMF.

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CONCLUSIONS Hierarchically structured composite materials were produced via a one-step self-organization on three hierarchical levels (i.e., by shearing of a dispersion consisting of Laponite and LC polymer). The structuring of Laponite nanoplatelets within the polymeric LC matrix is illustrated in Scheme 2. Gluing units Scheme 2. Illustration of Hierarchically Structured Composites Consisting of Laponite Nanoplatelets and LC Polymers

located on the polymer bind to the Laponite nanoplatelets, forming the glue between organic and inorganic components, which was demonstrated by AUC. An ordering of the polymer chains (chiral nematic phase, thus two hierarchical levels) and anisotropic nanoparticles (third level) is induced via liquidcrystal formation. Light microscopy revealed the ordering of the hierarchial composite films in the range of tens to hundreds of micrometers, and microtomography analysis showed the F

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Analytical Instrumentation and Methods. 1H NMR measurments were carried out at room temperature using a Bruker Avance III 400 operating at 399.79 MHz. CDCl3 (purchased from Deutero GmbH, Germany) was used as the solvent, and signals were referenced to δ = 7.26 ppm. Size exclusion chromatography (SEC) with simultaneous UV (270 nm) and RI detection was performed in NMP (+0.5 wt % LiBr) at 70 °C at a flow rate of 0.8 mL min−1 using two 300 × 8 mm2 PSS-GRAM columns (particle size, 7 μm; porosity, 102 and 103 Å). The calibration was done with polystyrene standards (PSS, Mainz, Germany). MALDI-TOF MS measurement was performed on a Bruker Microflex MALDI-TOF by using 10 μL of polymer precursor solution (2 mg mL−1 in THF), 10 μL of DCTB solution (10 mg mL−1 in THF), and 1 μL of sodium trifluoroacetate (0.1 mg mL−1 in acetone). Analytical ultracentrifugation (AUC) was carried out on a Beckman-Coulter XL-I using the Rayleigh interference optics and running the sample in selfbuild Ti double-sector cells at 25 °C. Light micrographs were taken in transmission mode with a Zeiss Axio Imager.M2m microscope with polarizers and a birefringence microscope (Abrio). The prerequiste for quantiative assignment, a constant film thickness on the submillimeter scale, was checked by means of a profilometer (Ambios XP2). SEM measurments were performed on a Zeiss CrossBeam 1540XB, and TEM images were recorded on a Zeiss Libra 120 microscope operating at 120 kV. SEM samples were sputtered with gold, TEM samples were embedded in Spurr resin and cured at 60 °C, and thin microtome slices were obtained with a Reichert Ultracut S microtome. SAXS measurements were carried out using a NanoSTAR diffractometer (Bruker AXS GmbH, Karlsruhe, Germany) with a Cu Kα X-ray source and two crossed Goebel mirrors, resulting in a wavelength of 0.154 nm and a beam size of approximately 400 μm diameter. The Bruker HiSTAR area detector was mounted 1050 or 260 mm from the sample, which was later calibrated using crystalline silver behenate powder. The intensity was determined as a function of the scattering vector q and corrected for background and dark current. Nanoindentation was performed using a Triboindenter TI950 nanoindenter (Hysitron, Minneapolis, MN, USA) using a Berkovich tip. Samples were embedded in PMMA and polished in the desired direction. The load function was set to a loading/unloading rate of 100 μN/s with a holding time at a peak load of 500 μN for 30 s. Data were collected in open-loop mode with 1026 points per indent. Load−displacement curves were analyzed for reduced modulus and hardness according to the Oliver and Pharr method.59 For microtomography, samples were scanned in tabletop laboratory microCT systems (Skyscan 1072 and 1172 models, Skyscan, Kontich, Belgium), revealing negligible contrast in our samples. Two of the samples were thus placed in the highresolution microtomography setup of the BAMline at BESSY-II (Berlin, Germany) using an energy of 10 keV.60,61 Similar to the labbased (conventional) tomography, 900 projections of the sample were obtained from 180° around the sample rotational axis. An effective pixel size of 0.88 μm was used. To obtain phase-contrast enhancement from the partially coherent X-ray source of the synchrotron, a propagation distance of 35 mm was used. The radiographs were concomitantly normalized and reconstructed by the backprojection method (PyHST, ESRF, Grenoble, France), rotated and cropped and 2D imaged (Image, NIH)62 or visualized in 3D (Skyscan CTvox 3D, Kontich, Belgium).

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected], [email protected]. Author Contributions

The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Nora Fiedler for help with polymer synthesis, Antje Völkel and Rose Rosenberg for AUC measurements, Lauretta Nejedli for microtome cutting of specimens for TEM measurements, the Proteomics Center (Konstanz), especially Dr. Andreas Marquardt, for MALDI-TOF MS measurements, and Ingrid Zenke for assistance with SAXS measurements. We thank HZB Berlin for access to BAMline for phase-contrast imaging and German Research Foundation DFG through Priority Programme 1420 “Biomimetic Materials Research: Functionality by Hierarchical Structuring of Materials” for financial support. P.Z. is grateful to the Berlin-Brandenburg Center for Regenerative Therapies (BCRT) for financial support.

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ASSOCIATED CONTENT

S Supporting Information *

Movie illustrating 3D view of the tomographically reconstructed volume of a composite consisting of Laponite nanoplatelets and LC gluing statistical copolymers P4 1:1 w/ w obtained after shearing by rotation. This material is available free of charge via the Internet at http://pubs.acs.org. G

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