Article pubs.acs.org/Macromolecules
Selective Molecular Permeability Induced by Glass Transition Dynamics of Semicrystalline Polymer Ultrathin Films Toshinori Fujie,‡,∥ Yuko Kawamoto,† Hiroki Haniuda,† Akihiro Saito,† Koki Kabata,† Yukio Honda,§ Eriko Ohmori,† Toru Asahi,†,‡,§ and Shinji Takeoka*,†,‡,§ †
Department of Life Science and Medical Bioscience, Graduate School of Advanced Science and Engineering, Waseda University, TWIns, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan ‡ European Biomedical Science Institute (EBSI), Organization for European Studies, Waseda University, Shinjuku-ku, Tokyo 162-8480, Japan § Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, Shinjuku-ku, Tokyo 162-0041, Japan ∥ WPI-Advanced Institute for Materials Research (WPI-AIMR), Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan S Supporting Information *
ABSTRACT: Most polymers solidify below a glass transition temperature (Tg), which is important for the fabrication of polymeric materials. The glass transition dynamics (GTD) of polymers alters their physical properties and therefore the range of applications suitable for the particular materials. In this regard, most GTD studies were oriented to the thermodynamics of amorphous polymer systems, while little studies were known for semicrystalline polymers. Here, we focus on the glassy and crystalline properties of semicrystalline polymers such as poly(L-lactic acid) (PLLA) and envisage to control the nanostructure of free-standing PLLA ultrathin films (referred as “PLLA nanosheets”), via thermodynamic rearrangement of polymer chains entangled in a quasi-two-dimensional interface during the GTD process. The annealing process on the PLLA nanosheets (106); (iii) unique interfacial and mechanical properties, such as tunable flexibility, noncovalent adhesiveness, and high transparency. From the structural viewpoint, a quasi-two-dimensional arrangement of polymer nanosheets could represent an ideal interface to mimic extracellular matrices in biological tissues, which comprise a well-organized permeable membrane that controls nutrient flux in living systems.21 Therefore, polymer nanosheets are regarded as a new category of quasi-two-dimensional soft materials. So far, various techniques to fabricate the free-standing polymer nanosheets have been introduced, including a simple spincoating method, a layer-by-layer (LbL) method, a Langmuir− Blodgett method with cross-linkable amphiphilic copolymers, and a sol−gel method with organic−inorganic interpenetrating networks.22−26 By choosing adequate fabrication methods, various kinds of free-standing nanosheets can be fabricated from various kinds of polymer sources including semicrystalline polymers. Poly(lactic acid) (PLA) (Tg ∼ 58 °C) is an aliphatic polyester made up of lactic acid (2-hydroxypropionic acid) building blocks.27 It has been widely studied because of its convenient production from lactic acid but also biocompatibility, biodegradability, and renewable resources, which make it a good candidate for valuable biomedical and environmental applications.28,29 In general, commercial PLAs are copolymers of poly(L-lactic acid) (PLLA) and poly(D,L-lactic acid) (PDLLA), which are produced from L-lactides and D,L-lactides, respectively. PLLA is known as a semicrystalline polymer, which is rubbery above Tg and becomes a glass below Tg. By contrast, PDLLA is an amorphous polymer without crystallinity. Although many researches are devoted to the study of physical properties of PLA materials, results are often found to be controversial due to its semicrystalline character.30,31 Thus, it is important to understand the thermodynamic behavior of PLA taking amorphous, glassy, and crystalline states into account since GTD in semicrystalline polymers drastically alter the nanostructure under the ultrathin region. In this study, we investigated the structural and mechancial properties of polymer nanosheets consisted of semicrystalline PLLA and amorphous PDLLA, respectively. Furthermore, we focused on the crystalline domains of free-standing PLLA nanosheets and envisaged to use them as molecular sieves of a filtration membrane. As far as we know, this is the first report to demonstrate the direct conversion of thermodynamic properties of semicrystalline polymers to the nanostructured properties such as selective molecular permeability without using conventional photolithographic microfabrication techniques or complicated molecular assembling processes.32−35
Article
EXPERIMENTAL SECTION
Materials. Silicon wafers (SiO2 substrates), purchased from KST World Co. (Fukui, Japan), were cut into an appropriate size (typically 2 cm × 2 cm) and immersed in a mixture of sulfuric acid/hydrogen peroxide (3/1) for 10 min before being extensively rinsed with deionized (DI) water (resistivity 18 MΩ cm). Two different isomers of PLA, PLLA (Mw 80 000−100 000; Tg 60−65 °C) and PDLLA (Mw 300 000−600 000; Tg 55 °C), were obtained from Polysciences Inc. (Warrington, PA). Poly(vinyl alcohol) (PVA, Mw 13 000−23 000) was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). An aqueous suspension of Au nanoparticles (AuNPs, 2.3 ± 0.5 nm in diameter) was purchased from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan). TEM grid (Cu, ϕ = 80 μm) was purchased from JEOL Ltd. (Tokyo, Japan). Unless stated otherwise, all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) and were used without further purification. Preparation and Crystallization of PLLA Nanosheets. Freestanding PLLA nanosheets were fabricated by spin-coating using a PVA sacrificial layer method (Figure S1). The film deposition process was achieved by the following steps: (1) an aqueous solution of PVA (1 wt %) was spincoated (Opticoat MS-A150, Mikasa Co. Ltd., Tokyo, Japan) on an SiO2 substrate at 4000 rpm for 20 s; (2) a dichloromethane solution of PLLA was spin-coated on the PVA layer (4000 rpm, 20 s); (3) the PLLA layered substrate was annealed at 80 °C for 2 h for crystallization; (4) the treated substrate was immersed in water in order to dissolve the PVA sacrificial layer, which allowed the release of the free-standing PLLA nanosheet. The PLLA nanosheets without annealing treatment were also prepared as control samples by omitting the process (3) and dried in vacuo overnight. The free-standing PLLA nanosheets were collected on the SiO2 substrate, PDMS slab, or transmembrane (Transwell Membrane, Corning Co., Inc., New York, NY) for materials characterization and/or molecular permeability tests. All the routines for the PLLA nanosheets fabrication were conducted in a clean room (class 10000) to avoid contamination on the sample surface. Structural Characterization. Thermal analysis of the PLLA nanosheet was conducted by differential scanning calorimetry (DSC) (Q200, TA Instruments, New Castle, DE). As sample preparation for a DSC measurement, we prepared around 20 sheets of free-standing PLLA nanosheets (without supporting substrates) in water and collected them in the air. Although the nanosheets were spontaneously corrugated during the drying process, we confirmed the overall structure of the nanosheets with free surfaces could be recovered once after dispersed in water again with freely suspended states. Then, we placed around 20 sheets of the collected nanosheets in a DSC pan until the total mass reached more than 2 mg. The measurement was performed at temperatures ranging between 40 and 200 °C with a heating rate of 10 °C/min under a dry nitrogen blanket. The software provided from the manufacture was used for analyzing the thermograms such as Tg, Tc, and Tm. As a control sample, we also performed DSC analyses for a melt crystallized bulk PLLA sample, annealed at 120 °C, 5 h by following the reported condition.31 Surface morphology was observed by an atomic force microscope (AFM) in tapping mode (MFP-3D-BIO, Asylum Research Co., Santa Barbara, CA) and by scanning electron microscopy. The scanning electron microscope (SEM) (HITACHI S-5500, Hitachi Co., Tokyo, Japan) was operated at an accelerating voltage of 5 kV with a platinum/palladium layer generated by an ion-sputtering coater (Hitachi E-1030, Hitachi Co; 15 mA, 10 s). The transmission electron microscope (TEM) (HITACHI H-7560, Hitachi Co., Tokyo, Japan) was operated at an accelerating voltage of 100 kV. Film thickness was measured by a surface profiler (α-step, KLA-Tencor Corp., San Jose, CA). The crystallization process of the PLLA nanosheet was evaluated by X-ray diffraction (XRD) (Rigaku Co., Tokyo, Japan). Monochromatized Cu Kα radiation was used for the experiment. The generator settings were 40 kV and 40 mA. The XRD patterns of each sample were resolved into crystalline (attributed to 16.5°) and amorphous regions (i.e., crystalline peaks and an amorphous halo, with a curve396
dx.doi.org/10.1021/ma302081e | Macromolecules 2013, 46, 395−402
Macromolecules
Article
Figure 1. Structural rearrangement of PLLA nanosheets induced by GTD: AFM images of the 60 nm PLLA nanosheet before (a) and after (b) the annealing process at 80 °C for 2 h. (c) A magnified AFM image of (b). fitting program).36 The crystalline contents in the different thickness of the PLLA nanosheets were normalized by estimating the ratio between the peak area of the nanosheets annealed at 80 °C (2 h) and 120 °C (6 h). Volume density of PLLA chains inside the nanosheet was evaluated using a quartz crystal microbalance (QCM) (Affinix QN, Initium Inc., Tokyo, Japan).37 Each free-standing PLLA nanosheet with different thickness was carefully collected on AT-cut QCM plates with an apparent frequency of 27 MHz (8 mm diameter of a quartz plate and an area of 4.9 mm2 of gold electrode) and dried in vacuo. Then, the amount of the adsorbed polymers (Δm) was measured by following the frequency shift of the QCM (ΔF) with time, using the following Sauerbrey’s equation38 ΔF = −
2F0 2 Δm ρq μq A
and for the PDMS equal to 0.33 and 0.50, respectively, in accordance with previous reports.39 Molecular Permeability Test. The molecular permeability through the PLLA nanosheet was investigated utilizing a Transwell membrane (TM) kit. The individual compartment of a 24-well microplate was isolated into inner and outer parts by a semipermeable transmembrane with pores of 8 μm in diameter. The free-standing PLLA nanosheet released in the distilled water was transferred and dried onto the transmembrane. All of the pores were densely covered with the PLLA nanosheet. Analytes of different molecular mass (rhodamine B (RhoB) 479 Da; vitamin B12 (VB12) 1355 Da; cytochrome c (CytC) 13.4 kDa; bovine serum albumin (BSA) 66.5 kDa) were dissolved in PBS and then added to the inner part of the well at a final concentration of 1 mM. In addition, we examined the separation of phenol red (354 Da) and IgG (150 kDa), which were dissolved in PBS at a final concentration of 75 mg/mL (212 mM) and 5 mg/mL (33.3 μM), respectively. The amount of each analyte in the bottom part of the well was continually monitored for a period of 24 h by UV−vis spectroscopy or by protein quantification assay (Bradford). Then, molecular permeability of each analyte through the PLLA nanosheet was evaluated as a function of cumulative release (%) from the inner part to the outer part of the well using eq 3
(1)
where F0 is the fundamental frequency of the QCM (27 × 10 Hz), A is the electrode area (4.9 mm2), ρq is the density of quartz (2.65 g cm−3), and μq is the shear modulus of quartz (2.95 × 1011 dyn cm−2). In this experiment, a frequency decrease of 1 Hz corresponded to a mass increase of 0.62 ng cm−2. Finally, the volume density was calculated by dividing the detected mass by the volume of the PLLA nanosheet attached to the electrode, in which the volume was calculated by multiplying the film thickness (obtained from surface profiler) by the electrode area. Mechanical Characterization. The mechanical properties of PLLA and PDLLA nanosheets were evaluated by means of “strain induced elastic buckling instability for mechanical measurement (SIEBIMM)”.22 The SIEBIMM test is based on the buckling metrology between an elastic substrate (such as polydimethylsiloxane: PDMS) and the nanosheet under compression or stretching, which allowed calculation of Young’s modulus of the nanosheet. The modulus is calculated by measuring the buckling wavelength of the nanosheet on a mechanically forced matrix. In practical, a free-standing PLLA nanosheet was collected from water onto a prestretched (∼3% strain of the original size) PDMS slab (3.0 cm × 3.0 cm and ∼2 mm thickness). Then, the prepared sample was dried in vacuo overnight prior to conducting the SIEBIMM test. After relaxation of the strain from the sample, a relative compression of the PLLA nanosheet was formed with a characteristic buckling pattern on the surface. The buckling pattern was immediately scanned by AFM, and the corresponding wavelength (λ) was measured from the AFM image and plotted as a function of film thickness. We tested at least six points using two different samples having the same thickness. Young’s modulus (EPLLA) for the PLLA nanosheets of different thickness (h) was individually obtained using eq 2 6
E PLLA = 3
E PDMS(1 − νPLLA 2) ⎛ λ ⎞3 ⎜ ⎟ ⎝ 2πh ⎠ 1 − νPLLA 2
[cumulative release (%)] = [Ct (mM)]/[C∞ (mM)] × 100 (3) where Ct is concentration of permeated molecule in the outer well at time t and C∞ is concentration of permeated molecule in the outer well at infinite time ∞. Then, flux was calculated from an initial gradient of the permeation curve using eq 4
[flux (mmol h−1 m−2)] = [Nt (mmol)]/[t (h) × A (m 2)]
(4)
where Nt is amount of permeated molecule in the outer well at time t (0.5 h) and A is the total area of the transmembrane pores (1.67 × 10−6 m2). Furthermore, AuNPs were infiltrated through the 60 nm thick PLLA nanosheets for the estimation of the possible mechanism of the molecular permeability. After the infiltration of AuNPs at 24 h, samples were dried in a desiccator and observed using TEM at an accelerating voltage of 100 kV.
■
RESULTS AND DISCUSSION The selective molecular permeability of the PLLA nanosheets was induced simply by annealing process at 80 °C, 2 h (PLLA (+)). However, such induction in molecular permeability was not observed for intact PLLA nanosheets (as prepared by spincoating) (PLLA (−)) or amorphous poly(D,L-lactic acid) (PDLLA) nanosheets (PDLLA (+)/(−)). Thus, first we focused on the thermodynamic property of PLLA nanosheets. Free-standing PLLA nanosheets with different thicknesses were prepared by spin-coating various concentrations of a PLLA solution on a SiO2 substrate covered with a PVA sacrificial layer (17 nm thick). Then, the substrate was subjected to the anneling process at 80 °C, and dissolution of the PVA layer in
(2)
where E and v represent Young’s modulus and Poisson’s ratio of the PLLA nanosheet and PDMS (i.e., EPDMS: 1.8 MPa), respectively. In this calculation, we assumed Poisson’s ratios for the PLLA nanosheets 397
dx.doi.org/10.1021/ma302081e | Macromolecules 2013, 46, 395−402
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DI water allowed the free-standing state of the PLLA nanosheet. In this regard, Torkelson et al. reported that glass transition of top free-surface layer in thin bilayer films was extremely sensitive to polymer species used in the underlayer when these two layers were consisted of analogous polymers (e.g., top: polystyrene; bottom: polystyrene).40 By contrast, in our study, PLLA was spin-coated on the dissimilar polymeric layer of PVA (i.e., top: PLLA; bottom: PVA). Thus, the cooperative segmental mobility of the PLLA layer should not be hindered by the PVA layer due to the immiscible nature of the two polymers. Therefore, influence of the PVA layer on the PLLA crystallization would be almost negligible. According to the thermal analysis by DSC measurements, both annealed and nonannealed PLLA (annealed at 80 °C, 2 h; defined as PLLA (+) and (−)) recorded similar Tg values of 58.3 and 58.0 °C, respectively (Figure S2). The similar Tg value (58.2 °C) was also confirmed for a melt-crystallized bulk PLLA sample (annealed at 120 °C, 5 h). Hence, PLLA nanosheets should be crystallized by annealing at 80 °C. In fact, atomic force microscope (AFM) images showed a remarkable morphological difference between PLLA (−) (Figure 1a) and PLLA (+) (Figure 1b), in which homogeneous distribution of grains attributed to PLLA crystals (∼100 nm in diameter) as well as microscopic apertures between crystals (∼100 nm in space) (Figure 1c) were clearly observed in PLLA (+). AFM images of PLLA nanosheets for different thicknesses (e.g., 201 and 531 nm) also showed marked differences between PLLA (−) and PLLA (+) (Figure S3). The observed differences are due to the mode of crystallization, in which PLLA crystals in PLLA (+) may accumulate as the film thickness increases. We also quantitatively analyzed the microscopic structure of the PLLA nanosheets by means of X-ray diffraction (XRD). A strong peak signal attributed to the PLLA crystals (16.5°) was detected in PLLA (+), whose peak area increased with film thickness (Figure 2a), while no signal was detected from PLLA (−) (Figure 2b). The relative crystalline content of PLLA (+) was estimated by normalizing the peak area at 80 °C with that at 120 °C. Our results reveal an incremental increase of crystalline content up to a thickness of 200 nm (Figure 2c). We also analyzed the volume density of the PLLA nanosheet collected on the QCM electrode. The denisty of the PLLA nanosheet kept almost constant values around 1.5 g cm−3 (Figure 2d). Based on this finding, PLLA chains are always packed inside the nanosheet at the same density regardless of the film thickness. Therefore, it is sugggested that crystallization behavior of semicrystalline polymer chains is determined by the confined space, that is, film thickness of the PLLA nanosheet. If we consider the microscopic mobility of polymer chains, PLLA chains in the thinnest nanosheets (
