Molecular Organization of Polylactides Immobilized on a Flat Surface

Jul 24, 2012 - Formation of single crystal arrays of polylactides (PLA) was found on a flat surface immobilized with poly(d-lactide) (PDLA) having dif...
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Molecular Organization of Polylactides Immobilized on a Flat Surface: Observation of Single Crystal Arrays of Homochiral and Stereocomplexed Polylactides Hajime Nakajima,† Maho Nakajima,† Tomoko Fujiwara,‡ Chan Woo Lee,§ Takashi Aoki,† and Yoshiharu Kimura*,† †

Department of Biobased Materials Science, Kyoto Institute of Technology Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan Department of Chemistry, University of Memphis, Memphis, Tennessee 38152, United States § Department of Innovative Industrial and Technology, Hoseo University Baebang-Myun, Asan, Chungnam 336-795, Korea ‡

S Supporting Information *

ABSTRACT: Formation of single crystal arrays of polylactides (PLA) was found on a flat surface immobilized with poly(D-lactide) (PDLA) having different chain lengths. Monoalkoxydimethylsilyl-terminated PDLAs were synthesized by hydrosilylation of (allyloxy)ethyl-terminated PDLAs and covalently immobilized onto silicon surfaces by a silicone coupling reaction in which the immobilization took place homogeneously through a single Si−O−Si bond. Atomic force microscopy (AMF) of the immobilized surfaces revealed that PDLA chains having Mn = 2400−7000 formed many projections (dots) on the surface of the polymer layers, each having a diameter of 30 nm and a height less than 1.0 nm with a narrow size distribution. When these surfaces were treated with free PDLAs and enantiomeric poly(L-lactide)s (PLLA) having similar chain lengths, nano-ordered arrangement of single crystallites arrays of homochiral (hc) and stereocomplexed (sc) PLAs were formed with ordering by the deposition of PDLA and PLLA chains on the surface-immobilized PDLA chains, respectively.

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surface.9 Tretinnikov et al. also reported surface initiated polymerization of L-lactide on a gold-coated glass surface.10 The surface PLLA layer on the gold formed stereocomplexed-PLA (sc-PLA) with free poly(D-lactide) PDLA, while the surface PLLA did not show any interaction with free PLLA. They proved surface immobilization of PLAs on the surface, but they did not study any surface morphologies of the PLAs layer. Akashi et al. reported a stepwise adsorption of enantiomeric PLLA and PDLA on a quartz surface to form a layer of sc-PLA by grafting to.11 Domb et al. reported indirect immobilization of PDLA chains on a surface pretreated with an aminoalkylsloxane derivative as well as the formation of several unique morphologies with the crystallization of the immobilized PDLA chains and the following stereocomplexation with the PLLA chains further deposited on the immobilized surface.12−14 They also observed the hetero stereocomplexation behavior between the immobilized PDLA chains and peptide molecules of L -leuprolide. In these studies, however, morphologies of PLAs could not be well controlled in nanoorder scale, probably because the surface immobilization of PDLA chains was not completely regulated. We recently

INTRODUCTION Polylactides (PLAs) have been utilized as functional bioabsorbable polymers for biomedical purposes such as tissue engineering, bone curing, and drug delivery.1−4 For their wider application, highly functional PLA materials ought to be developed by controlling their nano-ordered structure and morphology with a high level of precision. Another potential application of PLAs may be as surface modifiers having excellent biocompatibilities. The ordinary surface modification has been established by growing macromolecular chains on the surface by the “grafting from” method, in which a brushlike macromolecular layer can be formed.5,6 The alternative “grafting to” method has also been utilized for immobilizing preformed macromolecular chains by reaction with reactive sites on surface.7,8 The macromolecular chains thus grown and immobilized are often organized to form hierarchical structures such as Langmuir−Blodgett membranes and can be used as stimuli-responsive coatings, biocompatible scaffolds, and catalyst supports. Utilization of PLA polymers for the surface modification, however, has remained less accomplished until now because both the “grafting from” and “grafting to” reactions have been difficult. Previously, Langer et al. first reported surface-initiated polymerization of L-lactide on an inorganic substrate. They attained a controlled polymer layer of poly(L-lactide) (PLLA) thickness less than 500 nm on the © 2012 American Chemical Society

Received: May 18, 2012 Revised: July 4, 2012 Published: July 24, 2012 5993

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Scheme 1. First Deposition of mSi-PDLA on a Silicon Wafer and the Following Second Deposition of Free PDLA (Second hc Deposit) and Free PLLA (Second sc Deposit)

respectively, catalyzed by tin octoate at 120 °C with 1-hexanol as the initiator. These samples were named PLLA(X) and PDLA(Y), where Y denotes the number-average molecular weight (Mn) in kDa. Here, PDLA(3.4), PDLA(8.8), PLLA(2.7), PLLA(4.4), and PLLA(7.7) were prepared. A piece of silicon wafer (size 1 cm2) was treated with oxygen plasma using a plasma generator YHS-360 100 V (Sakigake Semiconductor, Kyoto, Japan) for 3 min. This generator is known to homogeneously oxidize the top surface only. Sakigake Semiconductor reported that the depth of the oxidation reaches 3 nm at maximum. After the plasma treatment, the silicon wafer was washed with deionized water containing a detergent (Scat 20x, Nacalai Tesque, Kyoto, Japan) in 5 wt % under sonication for 30 min at 40 °C and cleaned by dipping in distilled CH2Cl2 for 1 h. Synthesis of mSi-PDLA. The synthetic procedure of mSiPDLA(X), where X denotes Mn in kDa, was as reported previously.15 In short, an allyl-terminated PDLA (abbreviated as A-PDLA(X) where X = Mn in kDa) was first prepared by the ordinary ROP of D-lactide with EGMAE and tin octoate (1.0 mol % of EGME) as the initiator and catalyst, respectively. Then, a portion (3.00 g) of the resultant A-PDLA(2.0), for example, was dissolved in a CH2Cl2 (40 mL) and reacted with acetic anhydride (8.0 mmol) in the presence of pyridine (8.0 mmol) at room temperature to acetylate the terminal OH groups. The obtained acetylated product was solved in CH2Cl2 and reprecipitated in n-hexane. Then, 1.0 g of the acetylated product (abbreviated as A-ac-PDLA(X) where X = Mn in kDa) was reacted with EDMS (8.0 mmol) in the presence of Pt-DTS (0.01 mL) in refluxing CH2Cl2 (20 mL) for 12 h under a nitrogen atmosphere. Finally, the reaction system was thoroughly evaporated to obtain the mSi-PDLA(2.4) as the residue, which was preserved in a freezer under a nitrogen atmosphere. Here, three samples, mSi-PDLA(X) (X = 2.4, 3.3, and 7.0), were prepared. The structures of the A-ac-PDLA(7.4) and mSi-PDLA(7.0) were supported by their 1H NMR spectra (see Supporting Information). A-ac-PDLA(7.4) (quantitative recovery, Mn = 7400 Da, Mw/Mn = 1.10). 1H NMR (CDCl3): δ (ppm) = 5.80 (m, CH2CH−, 1H), 5.15 (m, CH2CH−, 2H), 5.08 (q, CH of the lactate unit, 1H), 4.28 (m, CH2−O, 2H), 4.00 (double triplet, CH2−CH2−, 2H), 3.63 (t, O−CH2−, 2H), 2.12 (s, −OCHCH3, 3H), 1.61 (d, CH3 of the lactate unit, 3H). mSi-PDLA(7.0) (quantitative recovery, Mn = 7000 Da, Mw/Mn = 1.15). 1H NMR (CDCl3): δ (ppm) = 5.17 (q, CH of the lactate unit), 4.28 (m, CH2−O), 3.63 (q, CH3−CH2−O), 3.62 (t, O−CH2−CH2), 3.41 (t, -CH2−O−), 2.13 (s, −OCCH3), 1.59 (d, CH3 of the lactate unit), 1.19 (t, CH3−CH2−O), 0.58 (m, Si−CH2), 0.12 (d, Si−CH3). Comparison of these 1H NMR spectra revealed that the olefinic proton signals (δ = 5.80 and 5.15) of A-ac-PDLA(7.4) are almost completely replaced by the signal (δ = 0.58 ppm) assigned to the Si− CH2 of mSi-PDLA(7.0) and that new signals assigned to −CH2O−Si

reported a new method for directly immobilizing PDLA chains on a surface using two types of silyl-terminated derivatives synthesized by terminal hydrosilylation of allyl-terminated PDLAs that were readily prepared by the simple ring-opening polymerization (ROP) of D-lactide with 2-(allyloxy)ethanol (ethylene glycol monoallyl ether: EGMAE) as the initiator.15 We found out that the monoethoxydimethylsilyl-terminated PDLAs (mSi-PDLA), monovalent in terms of silicone coupling reactivity, can be immobilized homogeneously through the single Si−O−Si bond on a surface, whereas the trivalent trimethoxysilyl-terminated PDLAs can give a heterogeneous morphology due to the intermolecular cross-linking reaction of the trialkoxysilane moieties. We therefore concluded the former derivatives are more useful in controlling the nano-ordered surface morphology of the immobilized PDLA chains. In this study, these mSi-PDLAs having different molecular weights are immobilized onto a plasma-treated silicon wafer (the first deposition) to analyze the changes in immobilization state with the chain length of the immobilized PDLAs. The immobilized PDLA chains are then allowed to interact with free PLLA and PDLA molecules in the following second deposition (Scheme 1) to study their surface crystallization mediated by the immobilized PDLA chains. The nano-ordered molecular structures created on the surface and the specific morphology changes with the homochiral (hc) and stereocomplex (sc) crystallization are well analyzed by atomic force microscopy (AFM) combined with Fourier-transform infrared spectroscopy (FT-IR) and quartz crystal microbalance (QCM) to disclose a unique formation of single crystal arrays of hc- and sc-PLAs that have never been reported before.

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EXPERIMENTAL SECTION

Materials. Both D- and L-lactide were supplied by Purac/CSM Biochemicals (Gorinchem, The Netherlands). Ethylene glycol monoallyl ether (EGMAE) was purchased from Tokyo Chemical Industries, Ltd. (Tokyo, Japan). Ethoxydimethylsilane (EDMS) and a solution of platinum(0)−1,3-divinyl-1,1,3,3-tetramethyldisiloxane complex (Pt−DTS) in xylene (ca. 2 wt % in Pt concentration) were purchased from Gelest (Morrisville, PA) and Sigma-Aldrich (Steinheim, Germany), respectively. Other chemicals (tin octoate, acetic anhydride, pyridine, and 1-hexanol) and solvents (toluene, dichloromethane (CH2Cl2), acetonitrile, methanol, and n-hexane) were purchased from Nacalai Tesque, Inc. (Kyoto, Japan), and distilled before use. The PLLA and PDLA samples used for the second deposition were synthesized by the ordinary ROP of L- and D-lactides, 5994

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Scheme 2. Synthetic Route to mSi-PDLA(X)

Table 1. Characteristics of mSi-PDLAs

a

sample

Mn(×103) (1H NMR)

Mn(GPC) (103 Da)

Mw/Mn

recovery (%)

conva (%)

Tg (°C)

Tc (°C)

Tm (°C)

mSi-PDLA(2.4) mSi-PDLA(3.3) mSi-PDLA(7.0)

2.4 3.3 7.0

3.5 4.0 7.7

1.09 1.10 1.15

60 >99 >99

89 93 94

48 52 54

84 86 84

148 158 158

Percent hydrosilylation of the allyl terminals.

and Si−CH3 of mSi-PDLA(7.0) appear at δ = 3.63 and 0.12 ppm. These data strongly support the hydrosilylated structure of mSiPDLA(7.0). The percent hydrosilylation was determined from the integral ratio of Si−CH2 signal (δ = 0.58) and the remaining small olefinic signal (δ = 6.0 ppm). Measurements. Gel permeation chromatography (GPC) was conducted on a Shimadzu analyzer system composed of a LC-10A pump and C-R7A plus chromatograph equipped with a refractive index detector. A set of two identical columns of a fixed pore size (50 nm, 10 × 250 mm2; Jordi Associates, Bellingham, MA) was used. The mobile phase was chloroform flowing at a rate of 1.0 mL/min. The numberaverage (Mn) and weight-average (Mw) molecular weights were calibrated with polystyrene standards. FT-IR spectra were measured on a Spectrum One spectrometer (Perkin-Elmer, Eden Prairie, MN) from 4000 to 500 cm−1 with a resolution of 4 cm−1 in wavenumber at room temperature. 300 MHz 1H NMR spectra were obtained using an AV-300 spectrometer (Bruker Biospin, Germany) in CDCl 3 containing 0.03 vol % tetramethylsilane (TMS) as the internal standard. Differential scanning calorimetry (DSC) was conducted on a Shimadzu DSC-50 thermal analyzer under a nitrogen flow of 20 mL/min at a heating rate of 10 °C/min for about 2.0 mg of sample sealed in an aluminum pan. AFM was conducted in dynamic force mode (tapping mode) using a Nanoscope IIIa scanning probe microscope (Veeco Instruments, Somerset, NJ). A commercially available silicon tip with a diameter of 8 nm and a spring constant of 30−40 N m and a single beam cantilever of 125 μm long was used in a resonance frequency range from 50 to 150 kHz. The size of this AFM tip was enough for analysis of height difference as low as 0.05 nm. X-ray photoelectron spectroscopy (XPS) was performed on a JPS9010MC/SP spectrometer (JEOL, Tokyo, Japan) with an Al Kα X-ray source at room temperature. A standard silicon sample was used to calibrate the binding energies of the Si-2P electrons of Si (99.2 eV) and SiO2 (103.9 eV). Water contact angle was measured by using a model CA-S150 contact angle meter (Kyowa Kaimen Kagaku Inc., Saitama, Japan) at room temperature by placing a drop of Milli-Q water (5 μL) on a substrate. An average value of five measurements was obtained. Ellipsometry was conducted on a DVA-FL ellipsometer (Mizojiri Optical Co., Ltd., Tokyo, Japan). A He−Ne laser of 632.8 nm in wavelength was injected at an incident angle of 70°. The following parameters were used to determine the layer thickness, where no, ns, and ks denote air, no = 1; silicon oxide, silane- and polymer-derived

layers, no = 1.46; silicon substrate, ns = 3.85 and ks = −0.02, respectively. Three to five measurements were done on different spots of each sample, and the data were averaged to determine the layer thickness. QCM analysis was performed with an Initium AffinixQ4 system (Initium, Inc., Tokyo, Japan). The system consisted of a QCM sensor made of AT-cut quartz crystal with gold electrodes having a 4.9 mm2 circle area, 11 mL reaction bath, oscillation circuit, and computer equipped with Windows XP. The analysis was done by applying oscillation at 27 MHz. Surface Immobilization. All procedures were carried out in a clean room. All apparatuses, such as vials, Pasteur pipettes, and volumetric cylinders, were cleaned by dipping in concentrated H2SO4 for 3 h, rinsing with Milli-Q water several times, and drying before use. A predetermined amount of mSi-PDLA(X) was dissolved in CH2Cl2 at a concentration of 0.02 g/mL. A plasma-treated silicon wafer (size: 1 cm2) was then immersed in this solution for 1 h, dried at room temperature (the first deposit), and washed with CH2Cl2 several times to remove the unreacted mSi-PDLA(X). During these processes, the wafer surface was always kept vertical to the horizontal plane. Subsequently, the wafer having the first deposit was immersed in a solution (0.02 g/mL) of either free-PDLA(Y) (the second hc deposit) or free-PLLA(Y) (the second sc deposit) for dip-coating and dried thoroughly in air with the wafer surface keeping vertical to the horizontal plane. The wafer having the dried second hc deposit was directly subjected to the surface analyses without further treatment. The wafer having the dried second sc deposit was further dipped in an acetonitrile several times to remove free PLLA(X) and dried again in air. Surface immobilization onto a QCM cell of quartz crystal equipped with a gold electrode was also performed in the same procedure as described before. In short, the surface of the quartz cell was first cleaned by dipping in a piranha solution (97% H2SO4/30% H2O2 = 3/1 vol/vol) for 5 min and rinsed with Milli-Q water 3 times. After drying the cell under a nitrogen atmosphere, the surface was activated by the aforementioned plasma treatment and used as a control. A solution (0.5 μL) of mSi-PDLA(X) in CH2Cl2 (1.0 mg/mL) was then applied to the plasma treated surface. The solvent was thoroughly removed in a nitrogen flow and washed by pouring CH2Cl2 (0.5 μL) 5 times to remove the unreacted mSi-PDLA(X) (the first deposition). Then, a solution (0.5 μL) of PLLA(X) in CH2Cl2 (1.0 mg/mL) was applied to the surface having the first deposit. The cell was finally dried in a 5995

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Figure 1. AFM height image of the surface of plasma-treated Si wafer.

Figure 2. AFM height images of the first deposits of (a) mSi-PDLA(2.4), (b) mSi-PDLA(3.3), and (c) mSi-PDLA(7.0).

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nitrogen flow for 5 h and washed with acetonitrile (0.5 μL) 5 times to remove the free PLLA molecules which had not been involved in the sc formation with the immobilized PDLA chains in the second sc deposition.

RESULTS AND DISCUSSION

Synthesis of mSi-PDLA(X). Scheme 2 shows the synthetic route to mSi-PDLA(X). Typical results of the synthesis of 5996

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mSi-PDLA(7.0), having the longest chain length (Figure 2c), showed a dot morphology where many projections, having a similar diameter of ca. 30 nm, were arranged in arrays. Each projection had a height difference below 1.0 nm as observed in the sectional image, whereas the layer thickness measured by the ellipsometry was 3.1 nm. The thickness of the polymer layer ought to be determined by the number and molecular weight of the immobilized polymer chains. Although the surface concentration of silanol groups is unknown, the fact that the thickness of the polymer layers increases with increasing the chain length of mSi-PDLA may support their efficient surface immobilization. However, the immobilized polymer chains ought to lean on the surface and likely prevent the next coming mSi-PDLA chains from grafting to the surface Si−OH groups in the vicinity. Consequently, the immobilization may not have taken place homogeneously on the surface with leaving many Si− OH groups unreacted (Scheme 3a). The immobilized polymer

mSi-PDLA(X) samples are summarized in Table 1 together with their thermal properties. Each mSi-PDLA(X) was isolated as slightly yellowish powders in high recovery (>99.0%). The slightly yellowish color of the products may be due to the Pt-DTD catalyst involved, although it should give no effect on the reactivity and properties of mSi-PDLA(X). The hydrosilylated structure of mSi-PDLA(X) was supported by their 1H NMR spectra as described in the Experimental Section. The percent hydrosilylation of the allyl terminals determined by the 1 H NMR spectra was in the range of 89−94%. Therefore, the produced mSi-PDLA(X) involved a small amount of unreacted A-ac-PDLA, which, however, can be washed from the surface of a silicon wafer after the immobilization reaction. The monodispersed nature of the mSi-PDLA(X) was confirmed by the Mw/Mn values. Their glass transition temperature (Tg), crystallization temperature (Tc), and melting temperature (Tm), determined by DSC, were around 50, 85, and 150 °C, respectively. The lower Tg and Tm values than those of the ordinary PDLA can be attributed to the oligomeric nature of mSi-PDLA(X)s. Surface Morphology of the Plasma-Treated Si Wafer. Prior to the surface immobilization of mSi-PDLA(X)s, the surface morphology of the plasma-treated Si wafer was analyzed by AFM, XPS, and contact angle. Figure 1 shows the AFM height image of a plasma-treated Si wafer. It revealed a height difference of only 0.2 nm, supporting the complete flatness of the surface. The XPS spectrum of the same plasma-treated Si wafer showed two peaks at binding energies at 99.2 eV (Si) and 103.9 eV (SiO2) in an Si/SiO2 intensity16,17 ratio of 10/5 in several spots. Since the ratio had been 10/1 before the plasmatreatment, it was confirmed that the surface oxidation took place homogeneously. The formation of hydrophilic Si−OH was also supported by the dramatic change in water contact angle from 80.0 ± 2.5° to 6.0 ± 1.0° before and after the plasma treatment, respectively. It was evident from these analyses that the flat silanol-functionalized surface had been formed. Surface Immobilization of mSi-PDLA(X) (the First Deposit). A plasma-treated silicon wafer was first dipped in a solution of mSi-PDLA(X) in CH2Cl2 for the first dip-coating. The ethoxysilyl terminals of mSi-PDLA(X) readily reacted with the surface silanols by the ordinary silicone coupling reaction, and the PDLA macromolecular chains were successfully immobilized on the surface via the “grafting to” mechanism. Figure 2 shows the AFM height images for the first deposits of the three mSi-PDLA(X) (X = 2.4, 3.3, and 7.0) on the silicon wafer surface. The deposit of the shortest mSi-PDLA(2.4) showed many small projections (dots) having different diameters of from several to 20 nm, with a height difference of less than 0.7 nm (Figure 2a). Each projection may be made by aggregation of the immobilized PDLA molecules. The thickness of the polymer layer was determined to be 1.2 nm by the ellipsometry, being slightly larger than the height difference of the sectional image along the line shown in the AFM image. This difference suggests that the projections should have been made on a condensed polymer layer of the immobilized PDLA chains, and a part of the immobilized chains are submerged in the polymer layer. The deposit of mSi-PDLA(3.3) having a medium chain length showed similar projections (Figure 2b). The average size of the projections was about 20−30 nm in diameter and 0.7 nm in maximum height difference by AFM sectional analysis. The average thickness of the surface layer of mSi-PDLA(3.3) determined by the ellipsometry was 2.5 nm, being much thicker than the height difference. The deposit of

Scheme 3. Chain Conformations of PLAs in the (a) First and (b) Second Deposits on the Si Surface

chains, in return, ought to fill the space between the grafting sites to form a polymer layer without crystallization (amorphous state). The segments on the top surface of this polymer layer retain high mobility as to generate the specific dot morphologies for minimizing the surface tension. In this case, the surface roughness ought to be much larger than that of the substrate surface as indicated by the AFM height differences of the first deposits (0.7−1.0 nm) and the substrate Si wafer (0.2 nm). Comparison of the layer thickness measured by the ellipsometry and the height difference determined by AFM revealed that in mSi-PDLA(3.4) ∼ 72% of the immobilized chains are submerged in the polymer layer and only 28% (0.7 nm in depth) are responsible for the morphology formation on the top surface. Second Deposition of PDLA on the Surface-Immobilized PDLA. Free PDLA polymers (PDLA(Y) where Y = Mn in kDa) were dip-coated on the first deposits of mSi-PDLA(X) obtained above. The resultant second hc deposits are denoted as mSi-PDLA(X)/PDLA(Y) where the X and Y have been adjusted to be almost identical. The as-prepared deposits were then annealed at 80 °C for 1 h. Figures 3a and 3b show the AFM height images of the second hc deposits of mSi-PDLA(3.3)/ PDLA(3.4) and mSi-PDLA(7.0)/PDLA(8.8), respectively, before annealing. They show similar undulated morphologies 5997

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Figure 3. AFM height images of the second hc deposits of mSi-PDLA(3.3)/PDLA(3.4) (a) before and (a′) after annealing and mSi-PDLA(7.0)/ PDLA(8.8), (b) before and (b′) after annealing.

where the height differences of the projections are 0.9 and 1.4 nm in Figures 3a and 3b, respectively. Compared with the corresponding first deposits, the second deposit mSiPDLA(3.3)/ PDLA(3.4) is flatter than the first deposit, whereas mSi-PDLA(7.0)/PDLA(8.8) is almost identical with the first deposit. However, their layer thicknesses determined by the ellipsometry are much thicker than those of the corresponding first deposits: 26 and 28 nm for the mSi-PDLA(3.3)/ PDLA(3.4) and mSi-PDLA(7.0)/PDLA(8.8), respectively. These results indicate that sedimentation of the free PDLA(Y)

is highly promoted on the PDLA chains immobilized on the surface. After the annealing, the surface morphologies of the second deposits were dramatically changed as shown in Figures 3a′ and 3b′. In mSi-PDLA(3.3)/PDLA(3.4), a large number of projections having a similar size (150 nm in diameter and 11 nm in height difference) were produced. The ellipsometry revealed that the average thickness of the layer increased by 5 nm compared with that before annealing. In PDLA(7.0)/PDLA(8.8) (Figure 3b′), on the other hand, many scaly disks of a larger size (500 nm in edge length and 25 nm 5998

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Figure 4. AFM height images of the second sc deposits of (a) mSi-PDLA(2.4)/PLLA(2.7), (b) mSi-PDLA(3.3)/PLLA(4.4), and (c) mSiPDLA(7.0)/PLLA(7.7).

(Y). In each of the second deposits, the actual thickness of the polymer layer increased and suggested that a certain amount of free-PLLA were immobilized on the original first deposits. The mSi-PDLA(2.4)/ PLLA(2.7) showed small projections scattered on the surface (Figure 4a). The sectional image clearly revealed a conic shape of the projections (100 nm in base diameter and 8 nm in height). Since the layer thickness determined by ellipsometry was 6.0 nm, it reasonably corresponds to the projection height. This fact suggests that most of the immobilized PDLA chains have interacted with the PLLA chains in a 1:1 ratio during the second deposition to form sc crystals. mSi-PDLA(3.3)/PLLA(4.4) (Figure 4b), on the other hand, showed large skewed tetrahedral projections covering the whole surface. Since this tetrahedral morphology is well correlated with that of single sc crystals,19,20 the immobilized PDLA and free PLLA chains have formed sc crystals without heat treatment. The actual height of the polymer layer determined by ellipsometry was 8 nm, which is 5 nm lower than the height difference (13 nm)

in height) were formed. Since each of these disks, having no boundary domain, showed a hexagonal or rhomboid shape resembling the single hc crystallites of PLLA reported previously,18 it can be reasonably attributed to the hc crystallites of PDLA(8.8) propagating in the direction of the (200) plane during the annealing. We think that the PDLA chains both in the first and the second hc deposits, having retained amorphous state, are involved in the crystallization during the annealing. Second sc Deposition of PLLA on the SurfaceImmobilized PDLA. Figure 4 shows the AFM height images of the second sc deposits of free PLLA(Y) on the first deposits of the mSi-PDLA(X) having similar Mn. After the dip-coating of PLLA(Y), the wafers were immersed in acetonitrile several times. Since acetonitrile is the poorest solvent of sc-PLA but good solvent of hc-PLA, washing with this solvent can remove the abundant PLLA molecules that are adsorbed on the surface without interacting with the immobilized PDLA. The second sc deposits thus obtained are denoted as mSi-PDLA(X)/PLLA5999

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the first deposit of mSi-PDLA(7.0) to the second deposit of PLLA(7.7) investigated by QCM was 1.02, also corresponding to their molecular weight ratio of 1.10. However, the longer PDLA chains from mSi-PDLA(7.0) may have decreased the initial amount of immobilization (643 ng/cm2) because of the repulsion between the macromolecular chains immobilized on the surface, and the amount of free PLLA(7.7) interacting with them became lower (710 ng/cm2) in the second deposition. The equal size of the crystallites of Figures 4b,c suggests that the equatorial crystal growth is equilibrated while the axial growth is limited. Crystallization of the Deposits. The crystalline nature of the first and second hc and the second sc deposits were analyzed by IR spectroscopy. Figure 6a shows a typical FT-IR

estimated from the AFM sectional analysis. This difference may not be so large considering that the latter one is for a top height of a specific projection. As discussed above, most of the immobilized PDLA chains in the first deposits are allowed to lie on the surface with chainfolding. After the second sc deposition, both of the immobilized PDLA chains and the inserted PLLA chains must stand up to have a specific arrangement for sc crystallization (Scheme 3b). Therefore, the thickness of the polymer layer of the second sc deposits becomes significantly larger than that imagined from the thickness of the first deposits and the amount of PLLA fixed for the sc formation. Since the immobilization of PDLA in the first deposition was not homogeneously performed, the sc crystallites have been generated in island forms where PDLA chains have been immobilized in relatively higher densities to make the heterogeneous state of the first deposits show up. Figure 5a shows the mass changes between the first deposit of mSi-PDLA(3.3) and the second deposit of free PLLA(4.4)

Figure 6. FT-IR spectra of the first deposit of mSi-PDLA(7.0) and the following second sc deposit of PLLA(4.4), (a) 1720−1800 cm−1 and (b) 880−1000 cm−1 as well as (c) the second hc deposits of PDLA(8.8) on the first deposit of mSi-PDLA(7.0) before and after annealing in the wavenumber region of 920−980 cm−1.

Figure 5. Frequency shifts in QCM (a) after the first deposition of mSi-PDLA(3.3) and the second sc deposition of free PLLA(4.4) and (b) after the first deposition of mSi-PDLA(7.0) and the second sc deposition of free PLLA(7.7).

spectra in the ν(CO) stretching region of the first deposit of mSi-PDLA(7.0) and the following second sc deposit of free PLLA(4.4). Since the second sc deposit exhibited the ν(CO) peak 4 cm−1 lower than that of the first deposit, the hydrogen bonding of OC− and −CH3 to form the sc crystals is supported.21 Figure 6b shows the FT-IR spectra in the lower wavenumber region. The first deposit exhibited very weak absorptions at 955 and 923 cm−1 that can be assigned to the coil and 103 helix forms of the PDLA macromolecular chains, respectively, whereas the second sc deposit exhibited absorptions at 955 and 917 cm−1 that can be assigned to the coil and 31 helix forms, respectively.22 The latter absorption is shown in 5 cm−1 higher than that reported for the ordinary 31 helical form. This deviation may be attributed to the single

investigated by QCM. It is evident that the amount of mSiPDLA(3.3) immobilized in the first deposition and the amount of free PLLA(4.4) adsorbed in the second sc deposition were 1130 and 1580 ng/cm2, respectively. Their weight ratio of 1.40 is almost identical to their molecular weight ratio, i.e., 1.33, strongly supporting the 1:1 chain interaction between the firstimmobilized PDLA and the second-absorbed PLLA to form the sc crystals. The slightly larger chain length of PLLA(4.4) may result in covering of the sc crystals with PLLA chains. The mSiPDLA(7.0)/PLLA(7.7) also showed a similar morphology consisting of tetrahedral projections whose height was rather lower (Figure 4c). As shown in Figure 5b, the mass ratio of 6000

dx.doi.org/10.1021/ma3010058 | Macromolecules 2012, 45, 5993−6001

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(5) Matyjaszewski, K.; Miller, P. K.; Shukla, N.; Immaraporn, B.; Gelman, A.; Luokala, B. B.; Siclovan, T. M.; Kickelbick, G.; Vallant, T.; Hoffmann, H.; Pakula, T. Macromolecules 1999, 32, 8716−8724. (6) Liu, Y.; Klep, V.; Zdyrko, B.; Luzinov, I. Langmuir 2004, 20, 6710−6718. (7) Zhu, A.; Zhang, M.; Wu, J.; Shen, J. Biomaterials 2002, 23, 4657− 4665. (8) Soellner, M. B.; Dickson, K. A.; Nilsson, B. L.; Raines, R. T. J. Am. Chem. Soc. 2003, 125, 11790−11791. (9) Choi, S. I.; Langer, R. Macromolecules 2001, 34, 5361−5363. (10) Tretinnikov, O. N.; Kato, K.; Iwata, H. Langmuir 2004, 20, 6748−6753. (11) Serizawa, T.; Yamashita, H.; Fujiwara, T.; Kimura, Y.; Akashi, M. Macromolecules 2001, 34, 1996−2001. (12) Domb, A. J.; Slager, J. Eur. J. Pharm. Biopharm. 2004, 58, 461− 469. (13) Domb, A. J.; Slager, J. Biomacromolecules 2003, 4, 1308−1315. (14) Domb, A. J.; Slager, J. Biomacromolecules 2003, 4, 1316−1320. (15) Nakajima, H.; Fujiwara, T.; Lee, C.-W.; Kimura, Y. Biomacromolecules 2011, 12, 4036−4043. (16) Ulgut, B.; Suzer, S. J. Phys. Chem. B 2003, 107, 2939−2943. (17) Ichimura, S.; Koike, K.; Kurokawa, A.; Nakamura, K.; Itoh, H. Surf. Interface Anal. 2000, 30, 497−501. (18) Fujita, M.; Doi, Y. Biomacromolecules 2003, 4, 1301−1307. (19) Brizzolara, D.; Cantow, H.-J.; Diederichs, K.; Keller, E.; Domb, A. J. Macromolecules 1996, 29, 191−197. (20) Cartier, L.; Okihara, T.; Lotz, B. Macromolecules 1997, 30, 6313−6322. (21) Zhang, J.; Sato, H.; Tsuji, H.; Noda, I.; Ozaki, Y. Macromolecules 2005, 38, 1822−1828. (22) Sawai, D.; Takahashi, K.; Sasashige, A.; Kanamoto, T.; Hyon, S.H. Macromolecules 2003, 36, 3601−3605.

crystalline state of the short PDLA chains or nano size effect. It is therefore supported that the highly crystalline state of sc-PLA is responsible for the generation of the tetrahedral morphology observed by AFM. Figure 6c compares the absorptions in low wavenumber of the second hc deposit of mSi-PDLA(7.0)/ PDLA(8.8) before and after annealing. Both deposits exhibited the carbonyl absorption at an identical wavenumber which is assigned to the 103 helical chain conformation of PDLA. The unannealed sample showed a clear absorption peak around 955 cm−1 due to the coil form of PDLA chains in the amorphous phase, as described above. After annealing, this absorption peak was replaced by the peak around 923 cm−1 that can be assigned to the 103 helical form, suggesting that both immobilized and adsorbed PDLA chains have crystallized into the ordinary α-form to produce the granular and scaly structures by annealing observed by AFM.

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CONCLUSION We succeeded in homogeneously immobilizing PDLA chains on a flat surface of plasma-treated Si wafer by the “grafting to” technique via siloxane bonding by using novel monoalkoxysilylterminated PDLAs. Under the terminal fixation, the immobilized PDLA chains were found to form the amorphous state having an undulation less than 1 nm in height difference in spite of different molecular lengths of the PDLA chains. On the PDLA-immobilized surfaces (the first deposit), free PDLA and PLLA polymers were dip-coated to form hc and sc crystallites with high surface density, respectively. It was evident that the immobilized PDLA and free PLAs can interact in molecular level in the whole surface to form nano-ordered single crystallites of hc- and sc-PLA in an array with significant thickness. This finding is the first report on the precisely nanoordered morphology control of PLAs on a flat surface and will shed light on how to control nano-ordered surface morphology with highly biocompatible PLA materials. The resultant PLA-immobilized surfaces should provide new functional substrates for controlled tissue engineering, cell cultivation, and drug delivery.

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ASSOCIATED CONTENT

S Supporting Information *

Enlarged 300 MHz 1H NMR spectra of (a) A-ac-PDLA(7.4) and (b) Si-PDLA(7.0). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the Kyoto Environmental Nanotechnology Cluster from the Ministry of Education, Culture, Sports, Science and Technology (MEXT). Sakigake Semiconductor and Purac/CSM Biochemicals are highly acknowledged for their support and helpful discussions.

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REFERENCES

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dx.doi.org/10.1021/ma3010058 | Macromolecules 2012, 45, 5993−6001