Enzymatic Degradation of Monolayer for Poly(lactide) Revealed by

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Enzymatic Degradation of Monolayer for Poly(lactide) Revealed by Real-Time Atomic Force Microscopy: Effects of Stereochemical Structure, Molecular Weight, and Molecular Branches on Hydrolysis Rates Keiji Numata,† Anna Finne-Wistrand,‡ Ann-Christine Albertsson,‡ Yoshiharu Doi,§ and Hideki Abe*,†,§ Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8502, Japan, Department of Fibre and Polymer Technology, School of Chemical Science and Engineering, Royal Institute of Technology, SE-100 44 Stockholm, Sweden, and Chemical Analysis Team, RIKEN Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan Received March 18, 2008; Revised Manuscript Received June 4, 2008

The influences of the stereochemical structure, the molecular weight, and the number of molecular branches for poly(lactide) (PLA) on enzymatic hydrolysis rates of PLA monolayers were studied by atomic force microscopy (AFM) and the Langmuir-Blodgett (LB) technique. Monolayers of six kinds of PLA with different molecular weights, stereochemical structure, and numbers of molecular branches were prepared by LB techniques and then characterized by AFM in air. The PLA molecules covered homogeneously with a silicon substrate and did not form lamellar crystals in the monolayer. We determined the initial hydrolysis rate of PLA monolayers in presence of proteinase K by volumetric analysis from the continuous AFM height images. The presence of D-lactyl unit reduced the hydrolysis rate of the monolayer. The hydrolysis rate for the linear PLLA samples increased with a decrease in the molecular weight. In contrast, the rates of erosion for branched PLLA monolayers were independent of the molecular weight of samples. The erosion rate of branched PLLA monolayers was found to be dependent on the average molecular weight of PLLA segment in branched molecules, not on the overall molecular weight of samples. From these results, furthermore, the hydrolysis mode of PLAs by proteinase K is discussed.

Introduction Poly(lactide) (PLA) is synthesized from either lactic acid or its cyclic dimer (lactide) and is a biobased, biocompatible and biodegradable thermoplastic. Recently, PLA has attracted much attention as an environmental friendly material. The biodegradability of PLA by various enzymes has been studied by many groups.1–9 In particular, the enzymatic degradation of poly(Llactide) (PLLA) and its copolymers by proteinase K from Tritirachium album has been reported in several articles.5–9 Reeve et al. carried out the degradation of a series of PLA stereocopolymers by proteinase K and they found that the enzyme preferentially degrades L-lactyl rather than D-lactyl units.5 In addition, it was reported that the enzymatic degradation preferentially occurs in the amorphous region of PLA.5 Cai et al. studied the effect of the crystallinity of PLA containing 96% L-lactyl and 4% D-lactyl units on the enzymatic degradation by proteinase K and concluded that the degradation rate increases with decreasing crystallinity (1996).6 Vert et al. performed the enzymatic degradation of films of PLA stereocopolymers by proteinase K and suggested that L-L, L-D, and D-L bonds are preferentially hydrolyzed over D-D bonds.7,8 The degradability of branched PLA has been studied by a couple of groups.9,10 Arvanitoyannis et al. synthesized starshaped PLA, using glycerol or sorbitol as co-initiator, and studied the enzymatic and alkaline hydrolyses of branched PLA * To whom correspondence should be addressed. Phone: +81-48-4678000. Fax: +81-48-462-4631. E-mail: [email protected]. † Tokyo Institute of Technology. ‡ Royal Institute of Technology. § RIKEN Institute.

(1995 and 1996).9,10 In our previous report, degradation rates of films for branched PLAs were found to be accelerated with an increase in the number as well as a decrease in the molecular weight of molecular branches.11 These reports have demonstrated that branched PLAs with higher number of branches (chain ends) and lower molecular weight PLA segment show higher degradability. The researches concerning hydrolyses of PLAs have indicated that the degradation behavior of PLA is always dependent on solid-state and surface properties of PLA.12–14 Therefore, the solid-state properties of PLA should be controlled and regulated to study the true hydrolysis rate of PLA molecules without the other effects. It is known that Langmuir-Blodgett (LB) technique regulates solid-state properties and structure of polymeric monolayer in nanometer scale. The solid-state properties of the monolayer prepared by LB technique are governed by surface pressure of polymer molecules at the air/water interface. Thus, the monolayer prepared by LB technique is well suitable to evaluate the hydrolysis rate in consideration of its solid-state properties. The hydrolysis of PLA monolayer prepared by LB technique has been studied in several publications.11–14 Ivanova et al. studied the enzymatic, basic, and acidic hydrolyses of PDLLA monolayer with a barostat surface balance and estimated the hydrolysis rate constant based on the decrease in surface area at constant surface pressure.15,16 They also visualized the polymeric monolayers before and after the hydrolysis reaction by atomic force microscopy (AFM) in air.15,16 Moreover, Lee et al. investigated the hydrolysis rate of the monolayer of PLA copolymers at the air/water interface by using a barostat surface balance and static AFM.17,18

10.1021/bm800281d CCC: $40.75  2008 American Chemical Society Published on Web 07/18/2008

Enzymatic Degradation of Branched PLA Monolayer

Biomacromolecules, Vol. 9, No. 8, 2008

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Table 1. Molecular Weights of the Samples Used in this Study sample PLLA-L PLLA-M PLLA-H PDLLA branched PLLA4 branched PLLA22

number-avg molecular weight (GPC), Mn polydispersity Tg, °C 3000 84000 700000 90000 13000 24000

1.6 1.4 1.6 1.8 1.9 1.1

36 58 60 55 46 53

The purpose of this study is to evaluate the influence of the molecular weight and number of the branches in addition to the stereochemical structures of PLA on the enzymatic hydrolysis rates by using real-time AFM and polymeric monolayer. From the time-sequential AFM height images of the hydrolysis processes of the monolayer, we estimate the enzymatic hydrolysis rate of the monolayers in nanometer scale and discuss the effects of the number and molecular weight of PLA branches on the hydrolysis rates.

Materials and Methods Materials. PLLA with a lower molecular weight (PLLA-L) was purchased from Nacalai Tesque and was used without further purification. PLLA with a middle molecular weight (PLLA-M), PLLA with a higher molecular weight (PLLA-H), and PDLLA purchased from Polyscience Inc. were used without further treatment. Branched PLLAs, PLLA initiated from pentaerythritol (molecular weight: 136) with 4 branches (branched PLLA4) and PLLA initiated from polyglycerin (number of units: 20, molecular weight: 1498) with 22 branches (branched PLLA22), were prepared by the method reported previously.11 All weight-average molecular weights (Mw) and polydispersities (Mw/Mn) were measured by gel permeation chromatography (GPC) system with polystyrene standards (Shodex Standard SM-105, 1.3 × 103 to 3.1 × 106). GPC measurement was performed using a Shimadzu 10A GPC system with joint columns of Shodex K-806 and K-802 at 40 °C. Chloroform was used as mobile phase at a flow rate of 0.8 mL/min, and a sample concentration was set to be 1.0 mg/mL. Shimadzu CLASS-VP software was used to process the data. All glass transition temperatures (Tg) were measured by differential scanning calorimetry (Perkin-Elmer Pyris 1) equipped with a cooling accessory. The samples (3 mg) were encapsulated in aluminum pans and heated from -50 to 200 °C at a rate of 20 °C/min and were maintained at 200 °C for 1 min. Subsequently, they were quenched to -100 °C at a rate of -200 °C/min and then were heated from -100 to 200 °C at a rate of 20 °C/min. The Tg was taken as the midpoint of the change in heat capacity during the second heating. Table 1 lists the molecular weight and Tg of the samples used in this study. Proteinase K from Tritirachium album was purchased from Roche (Germany) and used without purification. Preparation of Polymeric Monolayer. A monolayer film of PLA was prepared using the L-B Film Deposition System (Nippon Laser and Electronics Laboratory, Japan). A square silicon wafer (10 × 10 mm2) was used as solid support and substrate. PLA was dissolved in chloroform to a concentration of 0.1 mg/mL. All LB experiments were performed at 25 °C. The PLA chloroform solution (5.0 µL) was spread on a Milli-Q water surface. The solvent evaporation was allowed for 500 s. Monolayer compression was performed at a constant velocity of 10 mm/min to a given final surface pressure (0, 10, or 20 mN/m), and was transferred vertically at a constant speed of 3 mm/min. And then the PLA monolayer obtained on the silicon substrate was dried in air at 25 °C for 1 day. Fourier-Transform Infrared Reflection Absorption Spectroscopy (FTIR-RAS). FTIR-RAS measurements of polymeric monolayers on silicon substrates were carried out by means of JASCO FT/IR-615 equipped with RAS-400/H. FTIR-RAS spectra were recorded at a

Figure 1. Pressure-area isotherms of PLLA-H (A, dotted line), PDLLA (B, solid line), and branched PLLA22 monolayer (C, broken line) spread on the surface of water at 20 °C.

resolution 4 cm-1 under vacuum with a liquid-nitrogen-cooled mercury-cadmium-telluride (MCT(Hg1-XCdXTe)) detector. AFM Observations. The polymeric layers prepared by LB technique were observed by AFM (Seiko Instruments Inc. SPI3800/SPA 300HV) in air. A 400 µm long silicon cantilever with a spring constant of 15 N/m was used in dynamic force mode (tapping mode) AFM. A scan rate was set to 0.8 Hz. A light tapping force (set-point values ) 0.85) was applied for all AFM observations in this study to avoid damage to the polymeric layer by the cantilever tip. The enzymatic reaction process of the PLA monolayer was observed by real-time AFM in 0.1 M TrisHCl buffer solution (pH 8.5) containing proteinase K at 20 °C by the following the method reported previously.19–21 A 400 µm long silicon cantilever with a spring constant of 1.5 N/m was used in the real-time AFM observation. The other condition of the values in AFM measurement was the same as that in air. The PLA monolayer on the silicon substrate was fixed to the bottom of the reaction vessel (diameter: 25 mm, height: 5 mm) with double sided carbon adhesive sheets. The vessel with sample was set on the AFM scanner, and 1.0 mL of phosphate buffer solution was poured into the vessel. The monolayer before enzymatic reaction was observed in the buffer solution. And then the enzymatic reaction was initiated by the addition of concentrated enzyme solution into the vessel, resulting that a final concentration of enzyme solution was 10 µg/mL.

Results and Discussion Preparation of PLA Monolayer. Monolayer films for six types of PLA samples were prepared by LB techniques. Figure 1 shows the typical surface pressure-area isotherms for PLA monolayers. Each pressure-area isotherm of PLLA-H, PDLLA, and branched PLLA22 showed a plateau at around 5-15 mN/ m. After deposit on a silicon substrate, the morphologies of monolayers were characterized by AFM in air. Figure 2 shows the AFM height images of the monolayers for PLLA-H prepared at different surface pressure. At the surface pressure of 0 mN/ m, the layer was incompletely covered with PLLA-H molecules, and many apertures were present. As shown in Figure 2B, the homogeneous monolayer was formed at the surface pressure of 10 mN/m, and then the thickness of the layer was approximately 3 nm. From the morphologies of monolayer it seemed that the three-dimensional texture, for example, lamellar crystal, was not formed in PLLA-H monolayer. The AFM height images of PLLA-H layer prepared at 20 mN/m revealed a thick layer with thickness of around 6 nm. The layers with the different surface pressures of the other samples showed a similar

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Figure 2. AFM height images of the monolayer for PLLA-H with different surface pressure: 0 mN/m (A), 10 mN/m (B), and 20 mN/m (C). These images were obtained in air at 25 °C.

Figure 3. Continuous AFM height images of the enzymatic degradation process of monolayer for PLLA-M in a buffer solution containing 10 µg/mL of proteinase K at 20 °C.

manner as PLLA-H. In this study, we therefore prepared PLA monolayers covered with polymer molecules homogeneously at the surface pressure of 10 mN/m. On the basis of FTIR-RAS measurement, it was confirmed that the deposited layer on a silicon substrate was composed of PLA molecules (data not shown). Real-Time AFM Observations during Enzymatic Hydrolysis. Enzymatic hydrolysis process of the PLA monolayers was observed by real-time AFM in 0.1 M Tris-HCl buffer solution (pH 8.5) containing 10 µg/mL of proteinase K at 20 °C. Figure 3 shows the typical continuous AFM height images of the enzymatic hydrolysis of PLLA-M monolayer deposited on a silicon substrate. Before the degradation, the monolayer was covered with PLLA-M molecules homogeneously (Figure 3A). During the enzymatic reaction, the monolayer was eroded, and many apertures were formed in the monolayer. The thickness of monolayer was maintained around 3 nm throughout the enzymatic degradation reaction. No morphological change

was detected for all samples in the absence of proteinase K, indicating that the erosion of PLA molecules hardly took place by nonenzymatic hydrolysis during incubation for 2 h at 20 °C. Figure 4 shows the real-time AFM height images of monolayers for PLLA-H (A-C), PDLLA (D-F), and branched PLLA22 (G-I) during enzymatic degradation by proteinase K. As shown in Figure 4A-C, enzymatic erosion of PLLA-H monolayer gradually proceeded, and the aperture areas in monolayer increased with reaction time. However, the most part of the PLLA-H monolayer was remained after the enzymatic hydrolysis for 2 h. For the PDLLA monolayer, significant change in layer morphology was not detected during the first 2 h of enzymatic reaction. Only slight erosion could be detected after the degradation for 2 h. Apparent erosion was detected for the monolayer of branched PLLA22, and the rate of erosion was much faster than the other PLA monolayers. Lee et al. investigated the enzymatic degradation of PLA monolayer at the air/water interface by using a barostat surface



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Figure 4. Time-sequential AFM height images of the enzymatic degradation processes of monolayer for PLLA-H (A-C), PDLLA (D-F), and branched PLLA22 (G-I) in a buffer solution containing 10 µg/mL of proteinase K at 20 °C.

balance.17,18 They qualitatively evaluated the hydrolysis rate of PLA monolayer from the relative change in area occupied monolayer maintained at a constant surface pressure during degradation.17,18 In their study, the area ratio (A(t)/A(0), where A(0) and A(t) represent the area occupied by monolayer at time 0 and t, respectively) of PLLA (Mn ) 72000) monolayer was decreased to around 0.7 during enzymatic degradation for 200 min with 200 µg/mL of proteinase K at 20 °C.18 In this study, we carried out the enzymatic degradation of PLA monolayer deposited on a silicon substrate with 10 µg/mL of proteinase K at 20 °C. The A(t)/A(0) ratio of PLLA-M (Mn ) 84000) monolayer at degradation time of 100 min was around 0.75, and the value was higher than the reported value. This is because both samples differed in substrate and condition of polymer molecules. Enzymatic Hydrolysis Rates of PLA Monolayers. We quantitatively estimated the enzymatic hydrolysis rate of PLA monolayer by volumetric analysis from the real-time AFM height images. The changes in volume of the monolayers during enzymatic degradation were determined from AFM images and converted into the weight changes by considering the density of PLLA amorphous phase (1.248 g/cm3).22 Figure 5 demonstrates the weight loss of PLA monolayer normalized in an unit area during the degradation. It has been known that the enzymatic degradation of a solid polymer substrate is a surface erosion phenomenon, whose rate

Figure 5. Weight loss per unit area of monolayers for PLLA-L (closed circle), PLLA-M (circle), PLLA-H (opened circle), PDLLA (closed squire), branched PLLA4 (closed triangle), and branched PLLA22 (opened triangle) as a function of hydrolysis time during enzymatic degradations by proteinase K at 20 °C. The data in this graph were calculated from real-time AFM height images.

can be followed by weight loss, provided that the exposed sample area is taken into account as a normalization factor. When the polymer substrate is in film form, such area is commonly considered to be constant during the degradation process, until holes develop in the film. Under such situation,

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Table 2. Number Average Molecular Weight of PLA Segment and Initial Hydrolysis Rates of the Monolayers for PLAs by Proteinase Ka sample

theoretical number of hydroxyl chain ends

Mn of PLA segment

hydrolysis rate constant k, min-1

initial hydrolysis rate, mg/(min · cm2)

PLLA-L PLLA-M PLLA-H PDLLA branched PLLA4 branched PLLA22

1 1 1 1 4 22

3000 84000 700000 90000 3200b 1000b

21 × 10-3 6.0 × 10-3 1.9 × 10-3 0.25 × 10-3 27 × 10-3 56 × 10-3

79 × 10-7 21 × 10-7 6.2 × 10-7 1.9 × 10-7 108 × 10-7 370 × 10-7

a Determined from the real-time AFM observations. b These values were calculated by the following equation: (overall Mn - Mn of initiator)/number of chain ends.

linear plots of normalized weight loss vs time are obtained. In contrast, if the surface exposed to enzymatic attack decreases during the degradation process, as it seems to happen in the monolayers of this study, the weight loss rate normalized toward the initial area is obviously expected to decrease with increasing degradation time. Therefore, the enzymatic hydrolysis rate for PLA monolayers was evaluated from our experimental data by considering the change of the exposed surface area of sample. The hydrolysis rate should be relative to the exposed surface area of sample, which shows by the following equation:

dA(t) ⁄ dt ) -k · A(t)

(1)

where A(t) is exposed surface area at hydrolysis time t, k is the hydrolysis rate constant. The integration of dA(t)/dt at t also gives the following equation:

A(t) ) A(0) · exp(-k · t)

(2)

From the density of PLA (δ), the thickness of sample (s), and eq 2, the weight loss of the sample in a unit area is given as:

weight loss per unit area ) [W(0) - W(t)] ⁄ A(0) ) δs[A(0) - A(t)] ⁄ A(0) ) δs(1 - exp(-kt)) (3) where the weight of sample at time t is W(t), that is, W(0) is the initial weight before the enzymatic degradation. From the plot at the initial hydrolysis time, we determined the hydrolysis rate constant k as listed in Table 2. In addition, the approximation of t to 0 in eq 3 gives the initial hydrolysis rate R as follows:

R ) δsk

(t ≈ 0)

(4)

The initial hydrolysis rates obtained from eq 4 are listed in Table 2. Yamashita et al. have determined the enzymatic degradation rate of PLLA amorphous region in the presence of proteinase K by using a quartz crystal microbalance method and reported that the enzymatic degradation rates of amorphous region of PLLA at 25 °C were around 6.7 × 10-4 mg/(min · cm2) with 200 µg/mL of enzyme and around 1.1 × 10-4 mg/ (min · cm2) with 10 µg/mL of enzyme.23 In this study, the enzymatic degradation rate of the monolayer for PLLA-M (Mn ) 84000) was estimated to be 21 × 10-7 mg/(min · cm2) at 20 °C with 10 µg/mL of enzyme, and the value was much lower than the reported values of amorphous PLLA film whatever the difference of reaction temperature was considered. The following two possibilities can be considered to explain the difference of erosion rate between the film and the monolayer samples. One is due to the difference of the molecular situation between the film and the monolayer samples. Tsuji and Miyauchi have investigated the effect of the presence of crystalline regions on the enzymatic degradation rates of amorphous regions by using semicrystalline PLLA samples.24 They found that the degradation rates of PLLA amorphous regions in the semicrystalline PLLA samples were lower than those of completely amorphous PLLA sample.24 We used the

PLA monolayer samples deposited on silicon substrates to estimate the enzymatic degradability in this study. The mobility of PLA molecules in monolayer may be restricted by the presence of basal silicon substrate. In addition, nonlamellar crystallinity of PLA, which reduces the degradation rate, possibly exists in the monolayer. As a result, the enzymatic degradation rates of PLA monolayer obtained in this study revealed relatively smaller values. The other one is caused by the manner of adsorption of the enzyme onto the monolayer films. As reported by Yamashita et al., the adsorption of proteinase K onto the PLLA film surface was irreversible during degradation experiment, so that the enzyme molecules on the film surface could be observed directly by AFM.23 On the other hand, the enzyme molecules were not observed on the PLLA monolayer surface during real-time AFM imaging in this study. The enzyme molecules may easily detach from the surface of monolayer because of the loss of polymer layer as an anchoring scaffold through the degradation by themselves. As a result, the amount of enzyme molecules participating in the hydrolysis reaction of PLLA monolayer was restricted compared with the film surface. In this study, the hydrolysis rate for the linear PLLA samples increased with a decrease in the molecular weight. Tsuji and Miyauchi also performed the enzymatic hydrolysis of PLLA films with different molecular weights ranging from 76000480000 by proteinase K, and similar dependence of hydrolysis rate on molecular weight has been reported.24 These results suggest that the hydrolysis rate of linear PLLA by proteinase K is governed by the molecular weight of polymers. In contrast to the linear PLLAs, it seemed that the rate of erosion for branched PLLA monolayers was independent of the molecular weight of samples. Actually, the enzymatic erosion rate of PLLA22 monolayer was larger than that of PLLA4, although the overall molecular weight of PLLA22 (Mn ) 24000) was larger than that of PLLA4 (Mn ) 13000). Here, we deal with the length of PLLA segments in branched molecules. Roughly, the average molecular weight of PLLA segments in branched molecules can be calculated from overall molecular weight and the number of branches (see Table 2). The molecular weight of PLLA segments for branched PLLA4 and PLLA22 were 3200 and 1000, respectively. It is of interest to note that the molecular weight of linear PLLA-L (Mn ) 3000) and the average molecular weight of PLLA segments in branched PLLA4 were almost the same and that the erosion rates were very close between two monolayers. In addition, the monolayer of branched PLLA22 with the smallest PLLA segment revealed the fastest enzymatic erosion. These results indicate that the erosion rate of branched PLLA monolayers is dependent on the average molecular weight of PLLA segment in branched molecules, not on the overall molecular weight of samples. Hydrolysis Manner of PLA by Proteinase K. Proteinase K has been known as a family of serine protease, and the original


substrate of the enzyme is an L-alanyl residue in the protein.25,26 The enzyme is capable of hydrolyzing the ester bond of L-lactyl unit, which is the corresponding derivative of L-alanyl unit converted peptide group into ester group. The protein containing L-alanyl unit as an original substrate of proteinase K is a watersoluble compound, whereas the PLLA molecule is a waterinsoluble substrate. The chain end of PLLA molecules has higher mobility than the main-chain units on the surface of solid substrate. Therefore, it is predicted that the proteinase K preferentially hydrolyzes PLLA molecules from the chain end unit via exo-mode. It was recently reported that the erosion rate of PLLA molecules increased with a decrease in molecular weight of linear polymers.24 They also concluded that the enzymatic hydrolysis of PLA proceeded via both endo- and exochain scissions.24 As mentioned above, the linear PLLA-L and branched PLLA4 had similar segment length and revealed almost same erosion rate. Because the PLLA segments in branched samples in this study were propagated from the hydroxyl group of polyols as an initiator, each segment had the alkoxide ester chain end connected with polyols and free hydroxyl chain end. Hence, the exo-mode hydrolysis by proteinase K may proceed from the hydroxyl chain end of PLLA molecules. However, the rate constant of enzymatic hydrolysis of PLLA samples was not proportional to the inverse of molecular weight, as shown in Table 2. It has been confirmed that the proteinase K essentially has an endo-mode hydrolysis activity. We therefore consider that a random chain scission of PLLA molecules via endo-mode hydrolysis takes place in addition to exo-mode hydrolysis from the hydroxyl chain end. By the random hydrolysis reaction, newly chain ends are generated from PLLA molecules, and then the exo-mode hydrolysis starts from the generated chain end. In contrast, the overall erosion rate of PLLA molecules is enhanced by the occurrence of endo-mode hydrolysis. Especially for PLLA with high molecular weight, the enhancement effect by endomode hydrolysis may develop. As a result, the rate constant of enzymatic hydrolysis of PLLA samples was not proportional to the number of chain end of the initial state.

Conclusions This article is the first report investigating the effect of number of polymer chain ends of branched PLAs on the enzymatic hydrolysis behavior of the PLA monolayers. Combination of the LB technique and real-time AFM observations is a useful method for visualizing and understanding the hydrolysis behavior in nanometer scale of degradable polymers. Six types of PLA monolayers differed in the hydrolysis behavior with time, implying the effects of stereochemical structure, molecular weight, and number of molecular branch on the hydrolysis. By the volumetric analysis from the continuous AFM height images, we estimated the initial enzymatic hydrolysis rates in weight of the monolayers; for example, the enzymatic hydrolysis rate in weight of PLLA-M was 21 × 10-7 mg/(min · cm2). The presence of a D-lactyl unit reduced the hydrolysis rate in the monolayer. Additionally, the hydrolysis rate for the linear PLLA samples increased with a decrease in the molecular weight, whereas the rate of erosion for branched PLLA monolayers was independent of the molecular weight of samples. Actually, the


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enzymatic erosion rate of PLLA22 monolayer was 6 times larger than that of PLLA4, although the overall molecular weight of PLLA22 (Mn ) 24000) was larger than that of PLLA4 (Mn ) 13000). The length of PLLA segments in branched molecules were noted in our discussion, so that the erosion rate of branched PLLA monolayers is not dependent on the overall molecular weight but on the average molecular weight of PLLA segment in branched molecules of samples. Furthermore, the results of the enzymatic hydrolysis constant for linear and branched PLAs suggest that a random chain scission of PLA molecules via endomode hydrolysis takes place in addition to exo-mode hydrolysis. Acknowledgment. This work has been supported by a grant for Ecomolecular Science Research from RIKEN Institute and The Royal Institute of Technology and was completed while K.N. was a JSPS Research Fellow.

References and Notes (1) Williams, D. F. Eng. Med. 1981, 10, 5. (2) Ashley, S. L.; McGinity, J. W. Congr. Int. Technol. Pharm. 1989, 5, 195. (3) Fukuzaki, H.; Yoshida, M.; Asano, M.; Kumakura, M. Eur. Polym. J. 1989, 25, 1019. (4) Lim, H.-A.; Raku, T.; Tokiwa, Y. Macromol. Biosci. 2004, 4, 875. (5) Reeve, M. S.; McCarthy, S. P.; Downey, M. J.; Gross, R. A. Macromolecules 1994, 27, 825. (6) Cai, H.; Dave, V.; Gross, R. A.; McCarthy, S. P. J. Polym. Sci., Part B: Polym. Phys. 1996, 34, 2701. (7) Li, S.; Tenon, M.; Garreau, H.; Braud, C.; Vert, M. Polym. Degrad. Stab. 2000, 67, 85. (8) Li, S.; Girard, A.; Garreau, H.; Vert, M. Polym. Degrad. Stab. 2001, 71, 61. (9) Arvanitoyannis, I.; Nakayama, A.; Kawasaki, N.; Yamamoto, N. Polymer 1995, 36, 2947. (10) Arvanitoyannis, I.; Nakayama, A.; Psomiadou, E.; Kawasaki, N.; Yamamoto, N. Polymer 1996, 37, 651. (11) Numata, K.; Srivastava, R. K.; Finne-Wistrand, A.; Albertsson, A.C.; Doi, Y.; Abe, H. Biomacromolecules 2007, 8, 3115. (12) Tsuji, H.; Tezuka, Y.; Yamada, K. J. Polym. Sci., Part B: Polym. Phys. 2005, 7, 380. (13) Tsuji, H.; Ogiwara, M.; Saha, S. K.; Sakai, T. Biomacromolecules 2006, 7, 380. (14) Tsuji, H.; Nishikawa, M.; Osanai, Y.; Matsumura, S. Macromol. Rapid Commun. 2007, 28, 1651. (15) Ivanova, T. Z.; Panaiotov, I.; Boury, F.; Proust, J. E.; Verger, R. Colloid Polym. Sci. 1997, 275, 449. (16) Ivanova, T. Z.; Panaiotov, I.; Boury, F.; Proust, J. E.; Verger, R. Colloid. Surf., B 1997, 8, 217. (17) Lee, W.-K.; Gardella, J. A., Jr. Langmuir 2000, 16, 3401. (18) Lee, W.-K.; Iwata, T.; Gardella, J. A., Jr. Langmuir 2005, 21, 11180. (19) Numata, K.; Hirota, T.; Kikkawa, Y.; Tsuge, T.; Iwata, T.; Abe, H.; Doi, Y. Biomacromolecules 2004, 5, 2186. (20) Numata, K.; Kikkawa, Y.; Tsuge, T.; Iwata, T.; Doi, Y.; Abe, H. Biomacromolecules 2005, 6, 2008. (21) Numata, K.; Yamashita, K.; Fujita, M.; Tsuge, T.; Kasuya, K.; Iwata, T.; Doi, Y.; Abe, H. Biomacromolecules 2007, 8, 2276. (22) Fischer, E. W.; Hans, J. S.; Wegner, G. Kolloid Z. Z. Polym. 1973, 251, 980. (23) Yamashita, K.; Kikkawa, Y.; Kurokawa, K.; Doi, Y. Biomacromolecules 2005, 6, 850. (24) Tsuji, H.; Miyauchi, S. Biomacromolecules 2001, 2, 597. (25) Ebeling, W.; Hennrich, N.; Klockow, M.; Metz, H.; Orth, H. D.; Lang, H. Eur. J. Biochem. 1974, 47, 91. (26) Sweeney, P. J.; Walker, J. M. In Enzymes of Molecular Biology (Methods in Molecular Biology); Burrell, M. M. , Ed.; Humana Press: Totowa, NJ, 1993, Vol. 16, p 305.

BM800281D

Enzymatic Degradation of Monolayer for Poly(lactide) Revealed by

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