Article pubs.acs.org/Biomac
Calcium Phosphate Mineralization in Cellulose Derivative/ Poly(acrylic acid) Composites Having a Chiral Nematic Mesomorphic Structure Takuya Ogiwara,† Ayaka Katsumura,† Kazuki Sugimura,† Yoshikuni Teramoto,‡ and Yoshiyuki Nishio*,† †
Division of Forest and Biomaterials Science, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagimoto, Gifu 501-1193, Japan
‡
S Supporting Information *
ABSTRACT: Calcium phosphate mineralization was conducted by using polymer composites of liquid-crystalline (ethyl)cellulose (EC) or (hydroxypropyl)cellulose (HPC) with poly(acrylic acid) (PAA) as a scaffolding medium for the inorganic deposition. The EC/PAA and HPC/PAA samples were prepared in colored film form from EC and HPC lyotropic liquid crystals of left-handed and right-handed chiral nematics, respectively, by polymerization and cross-linking of acrylic acid as the main solvent component. The mineralization was allowed to proceed in a batchwise operation by soaking the liquidcrystalline films in an aqueous salt solution containing the relevant ions, Ca2+ and HPO42−. The calcium phosphatedeposited EC/PAA and HPC/PAA composites (weight gain, typically 15−25% and 6−11%, respectively) retained the chiral nematic organization of the respective original handedness but exhibited selective light-reflection of longer wavelengths relative to that of the corresponding nonmineralized samples. From X-ray diffraction and energy-dispersive X-ray spectroscopy measurements, it was deduced that the calcium and phosphorus were incorporated inside the polymer matrices in three forms: amorphous calcium phosphate, hydroxyapatite, and a certain complex of PAA-Ca2+. Dynamic mechanical analysis and thermogravimetry revealed that the inorganic hybridization remarkably enhanced the thermal and mechanical performance of the optically functionalized cellulosic/synthetic polymer composites; however, the effect was more drastic in the EC/PAA series rather than the HPC/PAA series, reflecting the difference in the deposited mineral amount between the two.
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INTRODUCTION Many cellulose derivatives as macromolecules and even the fragmented microfibrils of fibrous celluloses (i.e., cellulose nanocrystals CNCs) have a self-assembling character to form a liquid crystalline phase in a suitable solvent under adequate conditions;1−6 the mesogenic arrangement in the anisotropic phase is usually cholesteric or chiral nematic as synonym. In the stream where cellulose-based nanocomposites have become of great interest in cellulosic materials research,7−10 the use of such a mesomorphic assemblage of the molecular derivatives or CNCs will further expand the variety of the advanced nanomaterials showing high functionality and performance.6,11−14 This can be shortly exemplified by inorganic hybridization using a chiral nematic CNC liquid crystalline template, the representative inorganic component being silica.6,14−16 In this study, we attempt a calcific mineralization in polymer composite films imprinted with the chiral nematic mesomorphy of cellulose derivatives. Conventional cellulose ethers, (ethyl)cellulose (EC) and (hydroxypropyl)cellulose (HPC), are used as the parent substance providing the liquid crystalline © XXXX American Chemical Society
structure. The supramolecular structure can be made immobilized in polymer films or gels by polymerization of solvent monomer constituting the lyotropics of EC or HPC, as one of the authors (Y.N.) has demonstrated the examples formerly.17−20 A similar technique recently has been applied to create chiral nematic CNC/polymer composites that show mechanical high-performance21 or intriguing photonic properties,22 the latter including sensitive changes in iridescence of hydrogels in response to external stimuli such as solvent, pH, or temperature. The monomer used here is acrylic acid (AA), and therefore anionic polyelectrolyte, poly(acrylic acid) (PAA), is a network sustainer for the cellulosic liquid-crystalline architecture. A biomineral, calcium phosphate, is deposited in the liquid-crystalline hydrated films of EC/PAA and HPC/PAA by a partly biomimetic method. With regard to the mineralization of calcium phosphate in cellulosic matrices, there have been many investigations as Received: September 25, 2015 Revised: October 28, 2015
A
DOI: 10.1021/acs.biomac.5b01295 Biomacromolecules XXXX, XXX, XXX−XXX
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HPC/PAA Series. In past observations,34 HPC solutions in AA exhibited weak cholesteric iridescence over a broad range of wavelengths even at high concentrations of as much as 70 wt %. In view of the data, the solvent AA here was mixed with standard solvents, methanol and water, that lead to chiral nematics of HPC to improve the vividness in coloration at comparatively lower polymer concentrations; a similar technique has been used in the preparation of colored HPC composites with other vinyl polymers.19 In the present study, the lyotropic solvent AA/methanol/water was made at a proportion of 2:1:2 in weight content, and HMPPh and EDM were fed at an equal concentration of 1.0 wt % to the mixed solvent. Liquidcrystalline solutions of HPC were prepared at different polymer concentrations (60−75 wt %) in almost the same manner as that applied to the EC/AA system. An aliquot of each lyotropic HPC/ monomer mixed solution was replaced to another vial and centrifuged, with addition of a GA aqueous solution and a slight amount of hydrochloric acid that was a catalyst for cross-linking HPC side chains by the dialdehyde. The concentration of GA was adjusted to 4−5 wt % with respect to the total amount of the resulting respective solutions. The solutions were processed into a gelatinous film via photopolymerization in a similar way to that described for the synthesis of EC/PAA composite films. Following that, the gel films were dried at ambient temperature to evaporate away a large portion of methanol/ water from the samples. After removal of residual solvents and traces of unreacted reagents by washing with distilled water, followed by airdrying and subsequent vacuum drying at 25 °C, solid films of HPC/ PAA binary composition were produced. Mineralization Treatment. For both the EC/PAA and HPC/ PAA series, strips about 10 × 10 mm2 (or 20 × 5 mm2 for DMA measurements, etc.) cut from their respective as-prepared films were employed for mineralization experiments. The experimental standards were set up by reference to a method exploited by Iwatsubo et al.35,36 for biomimetic HAp mineralization in poly(vinyl alcohol) (PVA)/PAA hydrogels. Salt solutions containing 5.0 mM CaCl2, 3.0 mM (NH4)2HPO4, 140 mM NaCl, and a mM in the monomer unit of PAA (DP = 25) were prepared in basic pH conditions of 7.5−9.0 (using NaOH), the amount of PAA being varied between a = 0.17 (pH = 7.5) and ∼0.5 mM (pH = 9.0). The presence of this PAA prevented precipitation in the solution phase as ion server before and during mineralization by virtue of the polyelectrolyte effect of raising the precipitation threshold in concentration of source ions.36,37 A few strips of EC/PAA composites were soaked in 200 mL of the salt solution prepared initially at pH = 9.0, whereupon the system whole was thermostated at 30 °C in a bath for 5 days (unless otherwise specified), with exchange of the solution for fresh aliquots several times so that the pH was intermediate between 8.5 and 9. A similar soaking treatment was applied to films of the HPC/PAA series, but the salt solution was frequently renewed at pH = ∼7.4 because prolonged swelling of the samples at pH ≥ 8 often resulted in getting out of film shape. After the soaked films were taken out from the solution, they were rinsed in distilled water for 10 min, then air-dried, and subsequently vacuumdried at 40 °C for more than 12 h. Measurements. Selective light-reflection of mesomorphic samples were examined by visual inspection and by measurements of reflection bands with a UV−vis. spectrometer (Hitachi U-4100). Apparent circular dichroism (CD) spectra were also recorded for selected samples on a Jasco J-820DH spectropolarimeter to determine the handedness of the chiral nematic helical structure. These spectral measurements were conducted in an optical alignment of the normal incidence of light beam to the surface plane of each film sample. Refractive index measurements were carried out using an Abbé refractometer (Atago Co., Ltd., Type 2T) equipped with a rotatable polarizer mounted over the eyepiece. Wide-angle X-ray diffraction (WAXD) measurements were made on a Rigaku Ultima IV diffractometer in reflection mode. Nickel-filtered Cu Kα radiation was used at 40 kV and 40 mA. Diffraction intensity profiles were collected in a range of 2θ = 4−60°. Fracture surfaces of EC/PAA and HPC/PAA composites were observed by using a field emission scanning electron microscope (FE-SEM), Hitachi S-4800,
instantiated by recent activities23−27 employing bacterial cellulose (BC) hydrogels usually soaked in a simulated body fluid (SBF).28,29 In most of the examples aiming at biomedical applications, the hydroxyls of BC were negatively charged (-O−) or transformed into other anionic functional groups, or a carboxyl group-containing polymer was mixed with the fibrous cellulose. This may be regarded as inspired by the general view30,31 that such acidic groups would enhance heterogeneous nucleation of hydroxyapatite (HAp, Ca10(PO4)6(OH)2) and control the crystal growth, in analogy with HAp mineralization in collagen-based natural bone tissues.31,32 In the present work, the structural fixer PAA is an acidic polymer that can also play a role of ion absorber for calcification; therefore, the mineralization process would be possible in the inside of the liquidcrystalline cellulosic composites. The purpose of this paper is to describe the new preparation of inorganic-hybrid materials of cellulosics by calcium phosphate mineralization, the growth of which is spatially restricted within the mesomorphic networks of EC/PAA and HPC/PAA. Our major interest is dedicated to the variations following the mineralization of the composites, particularly in their chiral nematic helical structure and optical properties. Attention is also directed to the thermomechanical stability of the cellulosic composites as optical medium, in expectation of the improvement after the inorganic hybridization.
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EXPERIMENTAL SECTION
Materials. The EC sample used was purchased from Tokyo Kasei Kogyo Co., Ltd. (nominal viscosity, 90−110 cps for 5% solution in 80:20 toluene/ethanol at 25 °C): weight-average and number-average molar masses, Mw = 1.4 × 105 and Mn = 4.2 × 104, respectively (from GPC measurements); degree of substitution, DS = 2.50 (from 1H NMR measurement). The other cellulose derivative HPC was also of commercial origin (Tokyo Kasei Kogyo Co., Ltd., 6−10 cps for 2% solution in water at 20 °C): Mw = 1.5 × 105 and Mn = 5.7 × 104, respectively (from GPC measurements); DS = ∼1.5 and MS = ∼3.9 (from 1H and quantitative 13C NMR measurements),33 where MS denotes an average number of introduced hydroxypropyl groups per anhydroglucose residue. AA monomer (Nacalai Tesque, Inc.) was purified by distillation before use. A photopolymerization initiator, 2hydroxy-2-methylpropiophenone (HMPPh; Sigma-Aldrich), and cross-linking agents, ethylene dimethacrylate (EDM; Wako Pure Chemical Ind., Ltd.) and glutaraldehyde (GA) in 25% aqueous solution (Wako Pure Chemical Ind., Ltd.), were used without further purification. Calcium chloride dihydrate (CaCl2·2H2O) and diammonium hydrogen phosphate ((NH4)2HPO4) were used as ion sources for mineralization, a low-molecular-weight PAA (degree of polymerization (DP), ∼25) was used as a precipitation inhibitor in salt solution (see below), and other conventional reagents were purchased from Sigma-Aldrich or Nacalai Tesque, Inc. and used as received. Preparation of Liquid Crystalline Films. EC/PAA Series. EC solutions in AA were prepared in a polymer concentration range of 40−57 wt % by mixing weighed amounts of EC and AA in a lightblocked glass vial over a period of 2−3 weeks. The solvent AA contained HMPPh and EDM for polymerization, usually at concentrations of 1.0 and 0.5 wt %, respectively. In the mixing process, the sample vial was turned upside down and centrifuged at intervals for the purpose of accelerating the dissolution of EC. The EC/AA liquid crystals thus prepared were each sealed in a layer of solution between a Teflon plate and a transparent PET film by using another Teflon film as spacer, which provided a charging interspace of about 45 × 30 × 0.12 mm3. The sandwiched lyotropic samples were placed in a dark room at ∼22 °C for 2−5 days and then allowed to solidify via polymerization of AA at 25 °C by 2−3 h irradiation of UV light with an intensity maximum at 352 nm. The EC/PAA composite films thus synthesized were washed with distilled water, then dried at 40 °C under reduced pressure, and stored in a desiccator until used. B
DOI: 10.1021/acs.biomac.5b01295 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules after sputter coating with platinum. The morphologies were compared between before and after mineralization of selected film specimens of the two series of composites. For the mineralized samples, energydispersive X-ray (EDX) analysis was also conducted using an EDAX Genesis XM2 to see the distribution profiles of elements C, O, Ca, and P in the inside of the hybrid materials. Dynamic mechanical analysis (DMA) was conducted by using a Seiko DMS6100/EXSTAR6000 apparatus in tension mode. Film specimens of rectangular shape (20 × 5 mm2) were used for measurements of the temperature dependence of the dynamic storage modulus E′, loss modulus E″, and mechanical loss tangent tan δ. The measuring conditions were as follows: temperature range, 25−230 °C; heating rate, 2 °C/min.; oscillatory frequency, 10 Hz. Thermogravimetric analysis (TGA) was also carried out for 20 mg of film fragments with a Shimadzu TGA-51 apparatus in an atmosphere of nitrogen flow (50 mL/min); each sample (vacuum-dried at 110 °C in advance) was heated from 30 to 700 °C at a scanning rate of 5 °C/min.
Figure 1. Selective light-reflection spectra for EC(x)/PAA films: (a) x (wt % EC) = 45; (b) x = 47; (c) x = 49; (d) x = 52; (e) x = 54, detected by a UV−vis photometer. Insets provide photographs of visual appearance of the corresponding samples of a 10 mm square size.
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RESULTS AND DISCUSSION In the present study, swollen films of liquid crystalline EC/PAA and HPC/PAA composites are utilized as a mineralization field to deposit calcium phosphate, the PAA component being assumed to induce the nucleation. The ionic species taken in the gel matrix should be Ca2+ and HPO42−; the anion species is assured by the server condition, pH = 7.5−9.0 at which phosphoric acid (pKa1 = 2.2; pKa2 = 7.1; pKa3 = 12.2)28 dissociates dominantly in the divalent form of HPO42−. An ideal reaction involving the absorbed ions may be the HAp formation accompanying hydrolysis:36
shifts to the blue side with increasing EC concentration, indicating the negative dependence of the chiral nematic pitch (P) on the concentration. When colored EC/PAA films were immersed and swelled in the salt solution for mineralization, the colorations shifted to the side of longer wavelengths in the optical spectrum. The degree of swelling was generally small; for instance, a 10 mm square size of film strip expanded to 12−12.5 mm2 for EC(45)/ PAA, and to 11−11.5 mm2 for EC(55)/PAA. Apparently, these changes in size and color of the swollen films were completed within 2 days after starting the mineralizing treatment. However, if the samples were taken out of the salt solution in 2 or 3 days, the washed and dried ones imparted a reflection color intermediate in wavelength between the color in the untreated state and that in the swollen state. More than 4-daysoaking was required for the samples to gain their respective reflection color saturated at the longer wavelength, which was ascertained by following the λM position in spectrophotometry. Figure 2 illustrates selective light-reflection spectra (detected by CD spectropolarimetry) and visual appearance for two films of m-EC(x)/PAA (x = 55 and 50) obtained via 5-day-soaking treatment, and the data are compared with those in their respective initial states. As seen in Figure 2, panel a, the mEC(55)/PAA film is colored greenish blue, while the original appearance was tinged with violet. The other m-EC(50)/PAA (Figure 2b) is reddish, the tincture being changed from the original greenish appearance. Besides these examples, it was confirmed that an almost uncolored film of EC(57)/PAA turned bluish violet, and a red-colored film of EC(45)/PAA became uncolored, after they underwent the mineralization process. In a control experiment with a soaking solution free of CaCl2 and (NH4)2HPO4, similarly treated EC/PAA films regained an essentially original coloration after drying, although there was a color shift toward longer wavelengths during the swelling. From these observations, it is strongly suggested that minerals associated with calcium and phosphate were incorporated in the color-changed films, and this deposition was responsible for the irreversible change. Actually we obtained the following data of weight gain for m-EC(x)/PAA films: ∼16.5% (x = 55), ∼18% (x = 50), ∼23% (x = 45), and ∼30% (x = 40), the rate being elevated with increasing PAA content.
5Ca 2 + + 3HPO4 2 − + H 2O → Ca5(PO4 )3 (OH) + 4H+ (1)
Or stepwise, 3Ca 2 + + 2HPO4 2 − → Ca3(PO4 )2 + 2H+
(2a)
Ca3(PO4 )2 + 2Ca 2 + + HPO4 2 − + H 2O → Ca5(PO4 )3 (OH) + 2H+
(2b)
In what follows, some prominent effects of mineralization on the supramolecular structure and physical properties of the liquid-crystalline composite films will be described. For convenience, the two series of samples prepared in substantially binary polymer composition are designated as EC(x)/PAA and HPC(x)/PAA, where x denotes a weight percentage of the EC or HPC component in the respective composite films. In the HPC series, x was calculated by correcting for the quantity of methanol/water present in the starting liquid-crystalline solution. After mineralization treatment, the composite samples are encoded as m-EC(x)/PAA and m-HPC(x)/PAA with a discriminatory prefix, but without correction of the numeral x for the increment in inorganic content. Mineralization Effects in EC/PAA Mesomorphic Series. Color Variations of Composite Films. EC/PAA composites were successfully obtained in film form by polymerization of the monomer solvent constituting the EC/AA lyotropics. EC(x)/PAA films prepared at x = 43−55 wt % were clearly colored, and, with varying EC content, the colorations covered a sequence of spectrum from red (∼45 wt %) through orange (∼47 wt %), green (∼49 wt %), and blue (∼52 wt %) to violet (∼54 wt %), as evidenced in Figure 1. The figure includes the selective light-reflection spectra for selected film samples together with photographs of their visual appearance. It can be seen that the wavelength λM giving a reflectance maximum C
DOI: 10.1021/acs.biomac.5b01295 Biomacromolecules XXXX, XXX, XXX−XXX
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Figure 2. Selective light-reflection spectra for (a) m-EC(55)/PAA and (b) m-EC(50)/PAA measured by CD polarimetry and displayed in comparison with data for the respective nonmineralized states. Insets show photographs of visual appearance of the corresponding samples of a 9.5 mm square size.
Meso- and Nanostructures in Hybrid Materials. As exemplified in Figure 2, the EC/PAA series exhibited CD spectra characterized by a positive ellipticity curve, whether or not the composite films were mineralized. This indicates prevalence of a left-handed chiral nematic structure in each film, as in the case of EC lyotropics in AA.34 Thus, the handedness of the supramolecular helical arrangement in the liquid crystals was unchanged not only by the solvent polymerization, but also by the mineralization process. Quantification of the chiral nematic pitch P was made by using a relation,38 λM = ñP, where ñ is an average refractive index of the mesomorphic medium. For the composition of 55 wt % EC shown in Figure 2, panel a, P = 241 and 320 nm were evaluated before and after mineralization of the film, respectively, from the data: λM = 366 nm and ñ = 1.521 for EC(55)/PAA; and λM = 487 nm and ñ = 1.524 for m-EC(55)/ PAA. Similarly, we estimated P = 337 and 457 nm for EC(50)/ PAA and m-EC(50)/PAA, respectively, as to the other example given in Figure 2, panel b. Figure 3 shows FE-SEM images of fracture surface morphology for films of EC(55)/PAA (Figure 3a) and mEC(55)/PAA (Figure 3b). A well-developed, periodic layered architecture was observed in the inside of both samples. The periodicity in the lamellar structure was estimated to be ∼110 nm for EC(55)/PAA and 150−160 nm for m-EC(55)/PAA, and these values were roughly in agreement with the data of P/ 2 (= 120 and 160 nm) determined for the respective samples by optical measurements. The mineralization process usually rendered the subjected film thicker to some degree, and the increment was larger relative to the size change in the surface plane of the film. Actually, the film of m-EC(55)/PAA was ∼1.3-times thicker than that of EC(55)/PAA, in contrast with an extension of 1.1-times observed for the sides of the surface square. Probably, the deposition of mineral onto the chiral nematic stratums increased the layer spacing (d), and this mainly contributed to the elevation in P of the EC/PAA composite. The fracture surface of the mineralized sample m-EC(55)/ PAA was also examined by EDX measurements to make an element map therein. The result is shown in Figure 4, together with a low-magnification SEM image of the cross-section. As can be seen from the mappings of Ca and P, these elements were distributed widely throughout the inside of the film. Furthermore, EDX analysis provided numeric data about the allocation of four elements C, O, Ca, and P in the same section,
Figure 3. FE-SEM photographs of fracture surface morphology for (a) EC(55)/PAA and (b) m-EC(55)/PAA. The fracture surfaces were spattered with Pt.
as follows by atomic percentage (At %): C, 66.3%; O, 30.1%; Ca, 2.68%; P, 0.93%. Using the data, we can estimate that the source ions, Ca2+ and HPO42−, were charged at 13 wt % in total into the EC/PAA (55:45) matrix; this is equivalent to a 15% weight gain for the original polymer film and consistent with the actually weighed 16.5% gain. EDX analysis for a mineralized film of m-EC(40)/PAA indicated again the uniform distribution of Ca and P in the inside and offered the elemental allocation data (At %): C, 62.2%; O, 32.2%; Ca, 3.76%; P, 1.84%. Thereby, the weight gain attended by the mineralization is estimated at 26%, which is comparable to ∼30% measured by the weighing test. In relation to the discussion given in the following, it should be noted here that the ratio of [Ca]/[P] by At % is 2.88 for m-EC(55)/PAA and 2.04 for m-EC(40)/PAA, D
DOI: 10.1021/acs.biomac.5b01295 Biomacromolecules XXXX, XXX, XXX−XXX
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Figure 5. WAXD intensity profiles for various films of EC/PAA = 55:45 composition: (a) EC(55)/PAA (before mineralization), (b) mEC(55)/PAA (after mineralization); (c) p-m-EC(55)/PAA (after post-treatment with 1 M NaOH aq.) In data c, diffractions coming from crystalline HAp are marked with the corresponding (hkl) index.
mineralization. Accordingly, the hexagonal approximation would be no longer applicable to the low-angle diffraction signal, which results in the difficulty in precisely evaluating the structural parameters d and φ for m-EC(55)/PAA. Figure 5 includes additional WAXD data c for a sample designated as p-m-EC(55)/PAA. This sample was obtained by further swelling treatment of the mineralized sample mEC(55)/PAA with 1 M NaOH aq. for >1 h, followed by washing and drying. (The film was whitened and the coloration of greenish blue was faded by this post treatment, however.) We can see several diffraction peaks from crystalline HAp (a = b = 9.42 Å, c = 6.88 Å, γ = 120°)41 in the data c. This crystallization of HAp can be interpreted as being caused by alkali hydrolysis of a so-called amorphous calcium phosphate (ACP; roughly Ca3(PO4)2)42 or other less-ordered precursors42,43 of HAp, probably these being the major entity of the inorganic phase present in the m-EC(55)/PAA sample. The observation of calcium phosphate at such a very low HAp crystallinity is not a rare case of the biomimetic mineralization, rather familiar in related studies24−26,35,36 using polymer networks including BC gels. In the present case, however, the restricted spatial condition of the mineral deposition that would be prevalent between the chiral nematic layers may be the primary factor preventing the development of crystalline HAp. This is rationalized by the appearance of perceptible crystal diffractions of HAp in the WAXD data of the post-treated sample p-m-EC(55)/PAA. Additionally remarking, at the present time, we have no clear evidence of preferred orientation of the HAp component for the p-m-EC(55)/PAA sample as well as for the just mineralized one. As revealed by the EDX analysis, m-EC(x)/PAA samples usually indicated a calcium and phosphorus atomic composition of [Ca]/[P] > 2, easily exceeding the ratios expected for stoichiometric HAp (1.67) and ACP (roughly 1.5). This suggests that Ca2+ ions surplus to the mineralization are still trapped in the samples, probably captured in a complex form of −COO−---Ca2+---−OOC- by the absorbent PAA component. The formation was supported by IR measurements: a CO stretching band at 1720 cm−1 of carboxyl groups, a prominent signal detected for EC/PAA films before mineralization, disappeared after the mineralization, and, instead, carboxylate
Figure 4. (a) SEM image and EDX mappings of (b) Ca and (c) P for the fracture surface of m-EC(55)/PAA.
and both values are higher than a stoichiometric constant of 1.67 for HAp. WAXD intensity profiles of EC(55)/PAA and m-EC(55)/ PAA are shown in Figure 5. Before mineralization, the liquidcrystalline film of EC(55)/PAA gave a diffraction peak at 2θ = 9.6°, besides a diffuse scattering halo centered at 2θ ≈ 19° (data a in Figure 5). Using the low-angle data, and assuming the short-range order in the chiral nematic domain to be analyzable in terms of a hexagonal packing39,40 of mesogenic EC chains, we obtain d = 0.93 nm as a repeating distance in the stacking of nematic layers. Then, a twisting parameter φ defined by φ = 360°d/P is estimated to be 1.38° for the mesophase of P = 241 nm that was a data derived from CD measurements for EC(55)/PAA. After mineralization, as shown by data b in the figure, the sample m-EC(55)/PAA displayed an apparently noncrystalline WAXD pattern, which is dominated by two combined broad peaks with the maximum at 2θ ≈ 22° and 40°, respectively. Contrastively, the principal peak reflecting the nematic arrangement before mineralization was degenerated into a very weak signal at 2θ ≈ 9°. This degeneration may be ascribed to the anisotropic deformation of the film sample on E
DOI: 10.1021/acs.biomac.5b01295 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules signals were observed at 1575 cm−1 (asymmetric C−O stretching) and 1410 cm−1 (symmetric C−O stretching) for the mineralized films. Enhancement in Thermal and Thermomechanical Performance. DMA data obtained for EC(55)/PAA and mEC(55)/PAA are shown in Figure 6; the values of E′ (part a),
After mineralization, the m-EC(55)/PAA sample (Figure 6, solid lines) retained a high modulus E′ of >4 GPa at temperatures lower than 160 °C, and even the E′ lowering above the temperature was limited to a small extent. Correspondingly, the E″ data presented quite a feeble dispersion peak around 165 °C, which indicates that the micro-Brownian trunk chain motions of polymer molecules were made inactive, presumably, by a cement-like effect of calcium phosphate deposited in the composite. If a criterion of hard materials (based on polymer) is specified by the terms E′ ≥ 1.0 GPa and tan δ ≤ 0.1, the mineralized sample satisfied the requirements unless temperature was elevated to as high as 210 °C, whereas the nonmineralized one did only in a temperature range of ≤135 °C. Additionally remarking, such a drastic improvement in thermomechanical property as noted after mineralization was not realized in differently treated EC(x)/ PAA composites, designated as Ca2+-EC(x)/PAA or HPO42−EC(x)/PAA, which contained only either ion of Ca2+ and HPO42− involved in the mineralization (see Supporting Information, Figure S1). In Figure 7, TGM curves are compared between two samples, EC(55)/PAA and m-EC(55)/PAA. Each set of data is
Figure 6. Temperature dependence of (a) the storage modulus E′, (b) loss modulus E″, and (c) loss factor tan δ measured for film samples of EC(55)/PAA (dotted line in red), m-EC(55)/PAA (solid line in black), and EC as reference (broken line in blue).
Figure 7. TGA data for EC(55)/PAA and m-EC(55)/PAA, obtained in a gas flow of N2.
shown by plotting the residual weight as bulk against temperature in percentage relative to the weight at 100 °C. The starting temperature of polymer degradation, Td, defined by the onset method using a tangent of the weight loss curve, was estimated to be ∼255 °C for the nonmineralized EC(55)/ PAA, which yielded a char as residue of 17.3% at 700 °C. The basically bimodal weight loss curve of this sample is owing to the composition of two polymers different in degradation rate; unblended EC rapidly lost the mass of weight in a range of 300−400 °C, whereas the weight loss of PAA was relatively slow and observed over a wide range of 250−500 °C (data not shown). On the other hand, the temperature Td of the mineralized m-EC(55)/PAA sample was estimated at ∼325 °C, indicating that the heat resistance as a bulk material was heightened by ∼70 °C relative to the situation before mineralization. The weight residue at 700 °C (40.0%) of mEC(55)/PAA was also much higher than that (17.3%) of the nonmineralized composite. The difference of ∼22.5% exceeds the weight gain of 16.5% checked just after the mineralization treatment. This is explained as due to a synergistic effect of polymer-mineral hybridization; that is, the flame resistance (paralleling the resistance to pyrolysis) of the polymer matrix was possibly encouraged by the deposition of calcium
E″ (part b), and tan δ (part c) are plotted as a function of temperature to be compared between the two samples. For reference, the figure includes data of an isotropic EC film (Tg ≈ 128 °C in calorimetry) prepared from acetone solution. Regarding PAA, a DMA result obtained formerly for a solution-cast film (Tg ≈ 115 °C) is available;44 the profile is very similar to the EC data, but the primary α dispersion is located at a somewhat lower temperature compared with the EC’s (138 °C in E″). Before mineralization, the EC(55)/PAA composite (Figure 6, dotted lines) gave a principal dispersion centering 142 °C in the E″ curve; the modulus E′ seriously diminished in the temperature range of 140−180 °C but came into a plateau above 190 °C; and the tan δ curve produced an intensity maximum of 1.0 at 152 °C. The reference EC sample (Figure 6, broken lines) underwent a marked plastic flow above 150 °C, as evidenced by divergence of the tan δ curve, whereby the values of E′ and E″ at >170 °C were actually out of the measurement range. By comparison with this, it can be said that the EC(55)/ PAA composite has a better thermomechanical performance as a result of the locking-in of the mesomorphic ordered assembly of EC with cross-linked PAA. F
DOI: 10.1021/acs.biomac.5b01295 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules phosphate therein. In similar TGA experiments for Ca2+EC(55)/PAA and HPO42−-EC(55)/PAA, the thermal resistance of the composites was far behind the case with calcium phosphate, although a 3−4% increase in the weight residue at 700 °C of the pyrolysis was noted (see Supporting Information, Figure S2). Next, we examined thermal stability of the cholesteric colorations of mineralized films. A typical result is illustrated here on the comparative inspection for two film samples; one film of EC(55)/PAA was colored bluish violet, and the other of m-EC(55)/PAA was greenish blue, in visual appearance at room temperature (see Figure 2a). In CD measurements, the spectra of the two samples remained almost unchanged with a peak maximum at λM ≈ 367 nm (EC(55)/PAA) or 489 nm (mEC(55)/PAA) in the temperature limits of ≤110 °C permitted for the apparatus. Visual observations were made for the films put in a heating box where the internal temperature was raised at a rate of 2 °C/min. As shown in Figure 8, panel a, the
appearance followed a similar course of color change with time, but slowly over a period of more than 12 h. These color changes can be taken as a transient phenomenon incident to the process to an optically isotropic state of the polymer composite. Concerning the mineralized film of m-EC(55)/PAA (Figure 8b), a green hue rather than blue became more prominent above 165 °C, and then a green dominance over the film continued during heating from 170 to 210 °C. When the temperature reached 215 °C, the green color became pale and partly replaced by a brownish one due to a certain extent of molecular decomposition of polymers; however, the film was still in a fairly hard material. Above 215 °C, the sample rapidly lost the cholesteric coloration. It is interesting to find that the turning points 165 and 210 °C coincide with the temperature position of a feeble transition signal and the temperature limit as a hard material, respectively, specified for m-EC(55)/PAA in the DMA study. In another route of observation, a film strip of m-EC(55)/PAA was heated to 175 °C and cooled to room temperature at a rate of ∼1.5 °C/min. The cooled sample imparted a green color as it was at 175 °C and gave a CD spectrum with the peak maximum at λM = 507 nm. Even in the case where the cooling way was rapid quenching, the result was the same with a tolerance of 1 nm in λM. Thus, the heat treatment above 170 °C yielded a shift of ΔλM = ∼19 nm relative to λM ≈ 489 nm (at ≤110 °C) of the original mEC(55)/PAA film. This increase in λM, parallel to elevation in P, would originate from a slight expansion in volume of the film, this expansion accompanying the small transition (virtually local chain motions) around 165 °C detected by DMA. The volume gain at >170 °C seems to be readily fixed into the cooled film by obedience of the volume relaxation to a very slow kinetics in the mineral-deposited system. To sum up the major points about the coloring behavior of m-EC(55)/PAA, (1) the hybrid film can hold reflective coloration up to ∼210 °C; (2) the coloration of as-mineralized film is stable below 165 °C; (3) in the range of about 165−200 °C, the color at 160 °C shifts to the red side due to slight expansion in volume of the film, but the new color at a given temperature can be perpetuated in the sample by simple cooling. Such color control as mentioned in point 3 was assured by visual inspection using a few films of other EC/PAA compositions. Mineralization Effects in HPC/PAA Mesomorphic Series. Via polymerization of AA and evaporation of methanol/water, HPC(x)/PAA composite films were prepared from lyotropic liquid crystals of HPC in the solvent concoction. The solvent removing process was inevitably accompanied by shrinkage of the filmy sample as well as condensation of the HPC component, which gave rise to an appreciable decrease in the chiral nematic pitch P relative to the lyotropic’s. Vividly colored films of x = 83 (blue) to 81 (yellow green) were obtained from HPC solutions of ∼72 wt % (yellow to orange) to ∼66 wt % (red). Also, composite films of x = 80 (orange) to 78 (red) were made from about 65−62 wt % HPC solutions colored faint red. Figure 9 collects photographs of visual appearance of selected HPC/PAA films (80−83 wt % HPC) and compares the reflective colorations between before and after mineralization. We can see a modest extent of color shift following mineralization for any of the polymer compositions, the shift invariably directing toward longer wavelengths of the optical spectrum. In the mineralization process (pH ≈ 7.4), the
Figure 8. Changes in visual appearance of liquid-crystalline films (10 × 10 mm2) of (a) EC(55)/PAA and (b) m-EC(55)/PAA, observed on the heat treatment: →, heating at a rate of 2 °C/min; =>, isothermal annealing; --->, cooling at a rate of 1.5 °C/min (see text for discussion).
nonmineralized EC(55)/PAA film retained the initial coloration to 140 °C on heating. At this temperature, however, the film was already soft and partly adhered to a glass plate of underlay. On further heating to higher temperatures, the film color turned into bright blue at 150 °C and greenish blue at 155 °C, and it disappeared above 160 °C. Even in the case where the temperature was kept constant at 150 °C, the film G
DOI: 10.1021/acs.biomac.5b01295 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules
situation in the nonmineralized state (i.e., HPC(81)/PAA). In Figure 10, panel a, selective light-reflection spectra are displayed in both ways of UV−vis photometry (upper part) and CD polarimetry (bottom part). From the UV−vis spectra, λM = 548 and 646 nm were read off as a peak-top position for HPC(81)/PAA and m-HPC(81)/PAA, respectively, the λM values being consistent with the film colors observed before and after mineralization (see Figure 9). Using refractive index data, we calculated the chiral nematic pitch as P = 361 and 424 nm for HPC(81)/PAA (ñ = 1.518) and m-HPC(81)/PAA (ñ = 1.521), respectively. The CD measurements provided a negative ellipticity signal indicating development of the righthanded chiral nematic structure, whether or not the sample was mineralized; however, the intensity maximum was actually out of the measurable scale. Thus, the HPC/PAA chiral nematics stand opposite to the EC/PAA series in the handedness of helical arrangement. Figure 10, panel b shows FE-SEM images observed for fractured films of HPC(81)/PAA (left) and m-HPC(81)/PAA (right), both data revealing a periodic layered structure. The periodicity was, on average, ∼175 nm in HPC(81)/PAA and ∼205 nm in m-HPC(81)/PAA, the values being in good accordance with the data of P/2 = 180 and 212 nm determined above for the respective samples. The rise in P following mineralization represents an increase of 17%, lesser compared with the accustomed results of >30% for the EC/PAA series.
Figure 9. Visual appearance of liquid-crystalline films, compared between (a) HPC(x)/PAA (before mineralization) and (b) mHPC(x)/PAA (after mineralization) of x (wt % HPC) = 80−83.
swelling of the films concerned was controlled in a level of ∼1.2 times in the square edge and ∼1.5 times in the thickness by cross-linking of both polymer components by GA and EDM jointers; yet, the swelling extent was generally higher compared with that in the EC series described earlier. Nevertheless, the weight gain measured for the mineralized series, m-HPC(x)/ PAA, was at a lower rate of 6 to 11% for x = 83 to 78. This may be ascribed to the shortage of PAA content in the polymer composites. Figure 10 compiles data associated with the chiral nematic supramolecular structure of m-HPC(81)/PAA as a representative of the mineralized HPC series, in comparison with the
Figure 10. Collective data of (a) selective light-reflection spectra and (b) FE-SEM images of fracture surface morphology, compared between HPC(81)/PAA (not mineralized) and m-HPC(81)/PAA (mineralized). In panel a, spectral data of the two samples are displayed in both ways of UV−vis photometry (upper part) and CD polarimetry (bottom part). H
DOI: 10.1021/acs.biomac.5b01295 Biomacromolecules XXXX, XXX, XXX−XXX
Article
Biomacromolecules The deposition of minerals into m-HPC(x)/PAA composites was indistinguishable by ordinary SEM observations, but the EDX elemental mappings confirmed uniform distributions of Ca and P in the inside of the composite films. Regarding the internal allocation of C, O, Ca, and P, for example, we obtained the following At % data: 67.9 C, 29.4 O, 2.02 Ca, and 0.63 P for m-HPC(80)/PAA; 69.2 C, 29.2 O, 1.15 Ca, and 0.45 P for mHPC(84)/PAA. From these data, the weight gain attended by inorganics addition was assessed at ∼9% for m-HPC(80)/PAA and ∼5.5% for m-HPC(84)/PAA, consistent with the results of the weighing test. Values of [Ca]/[P] ratio, 3.20 and 2.55, calculated for these mineralized samples exceeded the stoichiometry (1.67) of HAp, however. This again indicates that an extra amount of Ca2+ ions remain without calcifying in the composites, and IR measurements suggested an electrostatic interaction of the surplus cations with the PAA COO− groups, as in the case of EC/PAA mineralized films. In WAXD measurements, it was also suggested that the mineral phase in the m-HPC(x)/PAA composites was made up of ACP and lessordered HAp, in view of the fact that no clear crystalline HAp appeared without intense alkaline post-treatment of the mineralized samples. Figure 11 collects results of DMA and TGA measurements carried out for film samples of HPC(80)/PAA and mHPC(80)/PAA. In Figure 11, panel a, temperature dependence of E′, E″, and tan δ is compared between the two samples. Looking over the DMA data for the nonmineralized HPC(80)/ PAA film, we can see two dispersions around 70 and 120 °C in positioning from the corresponding E″ peaks. With reference to the literature data for HPC45 and its relatives,46 the lowtemperature signal should be the primary α dispersion of HPC mixed with PAA. The other signal appearing above 100 °C may be associated with resistance of the cross-liked polymer networks against the translational chain slippage (fluidization).46 However, this sample was a substantially soft material after onset of the primary transition on heating, as can be seen from the observations of E′ < 1.0 GPa and tan δ > 0.1 already at 70 °C. Following mineralization, the dispersion around 70 °C degenerated, and the lowering of E′ and E″ above 120 °C became much smaller in amplitude, as evidenced by the corresponding data for m-HPC(80)/PAA in Figure 11, panel a; this mineralized sample can be taken as a hard material at temperatures lower than 115 °C, from readings of tan δ and E′ values. This modest improvement in thermomechanical property of the HPC/PAA composite may be reasonable, in consideration of a relatively low content (
