In Vitro Hydrolysis of Poly(l-lactide) Crystalline Residues as Extended

In Vitro Hydrolysis of Poly(l-lactide) Crystalline Residues as Extended-Chain .... Rosario Benavente , Marcela Lieblich , José Luis González-Carrasco ...
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Biomacromolecules 2004, 5, 1021-1028

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In Vitro Hydrolysis of Poly(L-lactide) Crystalline Residues as Extended-Chain Crystallites: II. Effects of Hydrolysis Temperature Hideto Tsuji* and Kensaku Ikarashi Department of Ecological Engineering, Faculty of Engineering, Toyohashi University of Technology, Tempaku-cho, Toyohashi, Aichi 441-8580, Japan Received December 11, 2003; Revised Manuscript Received February 6, 2004

The effects of hydrolysis temperature on the hydrolysis behavior and mechanism of poly(L-lactide) crystalline residues or extended-chain crystallites were investigated in phosphate-buffered solution (50-97 °C), using gel permeation chromatography and differential scanning calorimetry (DSC). The hydrolysis of the crystalline residues proceeded from their surface composed of very short chains with a free end along the chain direction, irrespective of hydrolysis temperature, but the hydrolysis from their lateral surface could not be traced. The activation energy of hydrolysis for the crystalline residues (extended-chain crystallites) was evaluated to be 18.0 kcal mol-1 (75.2 kJ mol-1). The monotonic melting temperature (Tm) and crystallinity decreases occurred after their initial very small increases, excluding the monotonic crystallinity decrease at 97 °C with no initial increase. The Tm decrease reflects the decreased thickness of the crystalline residues. The equilibrium Tm of the crystalline residues (extended-chain crystallites) was estimated to be 464.5-464.9 K. The free energy values for the surface composed of very short chains with a free end, which are neighboring the air (or nitrogen during DSC scanning), were calculated to be 55.6-56.4 erg cm-2 for heat of fusion per unit mass ) 135 J g-1. The obtained surface free energy values are significantly higher than that for the surface composed of folding chains, tie chains, and the chains with a free end, which are neighboring the same kind of amorphous chains (39.9 erg cm-2). Introduction Poly(L-lactide) [i.e., poly(L-lactic acid) (PLLA)] is attracting much attention because it is biodegradable, compostable, producible from renewable resources, and nontoxic to the human body and the environment.1-12 Moreover, PLLA has a high mechanical performance comparable with commercial polymers such as polystyrene and, therefore, is utilized as biomedical materials for tissue regeneration and matrixes for drug delivery systems as well as alternatives for commercial polymers. In our previous studies, we have prepared PLLA crystalline residues or extended-chain crystallites by high-temperature hydrolysis of crystallized PLLA films in phosphate-buffered solution (PBS) at 97 °C,13,14 in which the chains in the amorphous regions were completely removed (Figure 1), and their hydrolysis behavior and mechanism have been investigated in PBS at 37 °C for a long period up to 512 days.15 The latter study15 disclosed the following results: (1) The hydrolysis of the PLLA crystalline residues in PBS at 37 °C proceeded from their surface composed of very short chains with a free end along the chain direction, but the hydrolysis from their lateral surface could not be traced. (2) The average hydrolysis rates of the PLLA crystalline residues estimated from the changes of number-average and peak top molecular weights (Mn and Mt, respectively) were 5.31 and * To whom correspondence should be addressed. Phone: +81-532-446922. Fax: +81-532-44-6929. E-mail: [email protected].

Figure 1. Schematic representation for formation of PLLA crystalline residues (extended-chain crystallites; B) by hydrolysis of a PLLA crystallized film (A).

5.01 g mol-1 day-1, respectively. Such low hydrolysis rates strongly suggest that the PLLA crystalline residues can remain for a long period such as about 2000 days (ca. 5.5

10.1021/bm034523l CCC: $27.50 © 2004 American Chemical Society Published on Web 03/09/2004

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years) in the human body even after PLLA loses its functions as biomaterials. (3) The increased melting temperature (Tm) and crystallinity (Xc) and the decreased half widths of X-ray diffraction peaks during hydrolysis were attributed to the decreased lattice disorder. Here, the expected decrease of Tm, which reflects the decreased thickness of the PLLA crystalline residues, was not observed at 37 °C even when hydrolysis was continued for the long period of 512 days. Probably, at 37 °C a decrease in Tm due to the decreased thickness of crystalline residues was overcome by an increase in Tm due to the decreased lattice disorder. An enormously long period of hydrolysis should be required at 37 °C for the former factor to become dominant and, thereby, for the crystalline residues to give a lower Tm. At a higher hydrolysis temperature, a significant decrease in Tm is expected to occur in a shorter period. Moreover, as far as we are aware, activation energy of hydrolysis (∆Eh), equilibrium Tm (Tm0), and free energy for the surface composed of very short chains with a free end (σe) have not been reported for the PLLA crystalline residues or extended-chain crystallites, which can be evaluated from the hydrolysis rates obtained at different temperatures and times. The purposes of this study were to investigate the hydrolysis behavior and mechanism of PLLA crystalline residues (extended-chain crystallites) containing a trace amount of chains in an amorphous state at different temperatures exceeding 37 °C, to compare the hydrolysis behavior at different temperatures, and to obtain their ∆Eh, Tm0, and σe values. For these purposes, we prepared PLLA crystalline residues by high-temperature hydrolysis of crystallized PLLA films in PBS at 97 °C (Figure 1), and the obtained crystalline residues were further hydrolyzed in PBS at 50, 70, and 97 °C for the periods up to 256, 90, and 8 days, respectively. The hydrolyzed crystalline residues were investigated using gel permeation chromatography (GPC) and differential scanning calorimetry (DSC). Though no fungi were contained in the hydrolysis media in this study, the results obtained especially for the temperature range of 5070 °C, in which temperature range thermofiles have a high activity, must give basic and crucial information for designing composting processes that contain PLLA commercial products. Experimental Section Materials. The synthesis of PLLA16 and preparation of PLLA crystalline residues (extended-chain crystallites)13-15 were carried out according to our previous articles. PLLA films were prepared by solution casting using methylene chloride as a solvent from PLLA purified by reprecipitation. The as-cast films were dried in vacuo for at least 14 days. Each of the films was placed between two Teflon sheets and then sealed in a glass tube under a reduced pressure. The sealed PLLA films were melted at 200 °C for 5 min and then crystallized or annealed at 160 °C for 600 min, followed by quenching at 0 °C. The crystallization temperature 160 °C was selected because at this temperature PLLA films had thick crystalline regions compared with those prepared at lower temperatures.

Tsuji and Ikarashi

To obtain PLLA crystalline residues or to remove the PLLA chains in the amorphous regions, hydrolysis of PLLA films (18 mm × 30 mm × 100 µm) was performed in 100 mL of 0.15 M PBS (pH 7.4) at 97 °C for 40 h. After the hydrolysis, obtained crystalline residues were washed thoroughly with distilled water at room temperature, followed by drying under a reduced pressure for at least 14 days. Hydrolysis of Crystalline Residues. The hydrolysis of the PLLA crystalline residues (20 mg) was carried out in 100 mL of 0.15 M PBS (pH 7.4) at 50, 70, and 97 °C for the periods of time up to 256, 90, and 8 days, respectively. After the hydrolysis, the crystalline residues were washed thoroughly with distilled water at room temperature, followed by drying under a reduced pressure for at least 14 days. The distilled water used for the preparation of PBS and washing of the hydrolyzed crystalline residues was of HPLC grade (Nacalai Tesque, Inc., Kyoto, Japan). Measurements. Mn, the weight-average molecular weight (Mw), Mt, and the molecular weight distribution of the PLLA crystalline residues were evaluated in chloroform at 40 °C by a GPC system (Tosoh Co., Tokyo, Japan; refractive index monitor: RI-8020) with two TSK Gel columns (GMHXL) using polystyrene as a standard. Tm and the enthalpies of crystallization and melting (∆Hc and ∆Hm, respectively) of the PLLA crystalline residues were determined with a DT-50 differential scanning calorimeter (Shimadzu Co., Kyoto, Japan). The crystalline residues (sample weight about 3 mg) were heated from room temperature to 200 °C at a rate of 10 °C min-1 under a nitrogen gas flow at a rate of 50 mL min-1. Tm, ∆Hc, and ∆Hm were calibrated using tin, indium, and benzophenone as standards. The crystallinities (Xc’s) of the PLLA crystalline residues were evaluated according to the following equation:13-16 Xc (%) ) 100(∆Hc + ∆Hm)/∆Hm(100%)

(1)

where ∆Hm(100%) is the enthalpy of melting of the PLLA crystal having an infinite crystal thickness. We used a value 135 J g-1 reported by Miyata and Masuko as ∆Hm(100%).17 We did not use the values 93 and 100 J g-1 reported by Fischer et al.18 and Huang et al.,19 respectively, because Niejenhuis et al.20 and we13 observed ∆Hm values exceeding 100 J g-1. By definition, ∆Hc and ∆Hm are negative and positive, respectively. The characteristics and properties of the PLLA crystalline residues before and after the hydrolysis in PBS for 256 days at 50 °C, 90 (64) days at 70 °C, and 8 days at 97 °C are listed in Table 1. Results and Discussion GPC. Figure 2 shows the GPC curves of the PLLA crystalline residues (extended-chain crystallites) before the hydrolysis and after the hydrolysis in PBS for 256 days at 50 °C, 90 days at 70 °C, and 8 days at 97 °C. Evidently, the molecular weight distributions shifted as a whole to a lower molecular weight with hydrolysis time, irrespective of the hydrolysis temperature. This agrees well with the result at 37 °C15 and, therefore, reveals that in the temperature range of 50-97 °C the crystalline residues were hydrolyzed from

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Table 1. Characteristics and Properties of PLLA Crystalline Residues (Extended-Chain Crystallites) before and after the Hydrolysis in PBS at Different Temperatures hydrolysis conditions temperature (°C)

time (days)

Mn/103 (g mol-1)

Mt/103 (g mol-1)

Mw/Mn

Tm (°C)

∆Hc + ∆Hm (J g-1)

Xc (%)

50 70 97

0 256 90 8

10.2 2.74 2.55 4.80

13.9 4.39 3.54 5.61

1.23 1.50 1.32 1.15

181.5 164.9 172.2a 171.4

71.6 48.0 68.6a 53.1

53.0 35.6 50.8a 39.3

a

The DSC data are for the specimen hydrolyzed for 64 days not for that hydrolyzed for 90 days, because of a small amount of specimen at 90 days.

Figure 2. GPC curves of PLLA crystalline residues (extended-chain crystallites) before hydrolysis (A) and after hydrolysis in PBS for 256 days at 50 °C (B), 90 days at 70 °C (C), and 8 days at 97°C (D).

their surface composed of very short chains with a free end along the chain direction, irrespective of hydrolysis temperature. Chen and Gardella indicated that the water-soluble oligomers with molecular weights up to 2000 g mol-1 elute from a mother PLLA film even at the first stage of hydrolysis.21 However, in our case it is expected that the ester groups neighboring the chain ends were cleaved step by step and the formed L-lactic acid or its water-soluble oligomers with relatively small molecular weights were released from the surface because water molecules are inhibited to diffuse into the crystalline regions. The hydrolysis of the crystalline residues may have occurred from the lateral surface but could not be traced by GPC probably because of a trace amount of hydrolyzed chains remaining there or a much smaller area of the lateral surface compared with that of the surface composed of very short chains with a free end. The Mn and Mw/Mn of the PLLA crystalline residues are plotted in Figure 3 as a function of hydrolysis time. The Mn of the crystalline residues decreased monotonically and linearly with hydrolysis time, and the decrease rate was higher at a higher temperature. The percentage remaining values of Mn at 256 days (50 °C), 90 days (70 °C), and 8 days (97 °C) were 22.0, 25.5, and 46.8%, respectively. This means that 78.0, 74.5, and 53.2% of the PLLA chains were removed as L-lactic acid or water-soluble oligomers from the surface composed of very short chains with a free end. At the initial stage of hydrolysis, the Mw/Mn values of the crystalline residues were constant around 1.2, whereas at the late stage of hydrolysis when their Mn decreased below 4000,

Figure 3. Mn (a) and Mw/Mn (b) of PLLA crystalline residues (extended-chain crystallites) hydrolyzed at different temperatures as a function of hydrolysis time.

the Mw/Mn of the crystalline residues increased to 1.50 at 256 days (50 °C) and to 1.32 at 90 days (70 °C). This is not attributed to the hydrolysis from the lateral surface but may be due to the decreased molecular weight of the specimens at the late stage, which seemingly increased the Mw/Mn values of the crystalline residues. In other words, at a low molecular weight, a deviation of molecular weight of each molecule from Mn relative to Mn becomes large even if its absolute deviation value remains unchanged. The average hydrolysis rates estimated from the changes in Mn assuming that linear decreases of Mn for the periods

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Figure 4. Arrhenius plot of the hydrolysis rate constant [k(T)] of PLLA crystalline residues (extended-chain crystallites).

studied here were 28, 86, and 710 g mol-1 day-1 at 50, 70, and 97 °C, respectively. This assumption means that the hydrolysis rates are constant irrespective of the molecular weights of the PLLA crystalline residues and is justified at least in the Mn range studied here (2 × 103 to 1 × 104 g mol-1). The overall periods required for the crystalline residues having an initial Mn of 1.02 × 104 g mol-1 to become a molecular weight of L-lactic acid, 90 g mol-1, were evaluated to be 361, 118, and 14 days at 50, 70, and 97 °C, respectively. It should be noted that these periods depend on the molecular weight of the crystalline residues before hydrolysis and that the increase in Mw/Mn at the late stage of hydrolysis will affect the degradation rate estimated by Mn. The finding here indicates that the crystalline residues will remain for a long period even after PLLA loses its functions as materials. In contrast, Joziasse et al. estimated the in vivo resorption time of high-molecular-weight aspolymerized PLLA assuming the exponential decrease of molecular weight to be 40-50 years.22 Activation Energy for Hydrolysis. The hydrolysis rate of the PLLA crystalline residues is expected to depend on solely the hydrolysis temperature but not on the chain length or molecular weight of the crystalline residues. Thereby, the hydrolysis rate can be expressed only by the hydrolysis rate constant as a function of temperature (T), that is, hydrolysis rate ) k(T). The Arrhenius plot of the obtained k(T) or hydrolysis rate is presented in Figure 4, which gives ∆Eh for the crystalline residues. In this figure, k(T) at 37 °C reported in the previous study is also included.15 The estimated ∆Eh value of the PLLA crystalline residues (extended-chain crystallites) at the temperature range of 3797 °C was 18.0 kcal mol-1 (75.2 kJ mol-1), which is significantly higher than 12.2 kcal mol-1 obtained by ourselves for PLLA in the melt at the temperature range of 180-250 °C23 but is slightly lower than 20.0 and 19.9 kcal mol-1 reported by Makino et al. for PLLA and poly(DLlactide) [i.e., poly(DL-lactic acid) (PDLLA)] microspheres, respectively, as a solid at the temperature range (21-45 °C) below the glass transition temperature (Tg).24

Figure 5. DSC thermograms of PLLA crystalline residues (extendedchain crystallites) before hydrolysis (A) and after hydrolysis in PBS for 256 days at 50 °C (B), 64 days at 70 °C (C), and 8 days at 97 °C (D).

The rather small ∆Eh value of the PLLA crystalline residues compared with those of PDLLA and PLLA microspheres in a solid state containing a large amount of highly hydrolyzable amorphous chains can be ascribed to the easy access of water molecules to the ester groups neighboring the surface of the crystalline residues rather than to those of PDLLA and PLLA microspheres in a solid state. In the former case, water molecules can approach ester groups directly from outside of the crystalline residues, while in the latter case water molecules should diffuse into polymer matrixes to reach the ester groups. It is interesting to note that the k(T) shows a monotonic exponential decrease with T-1 for the temperature ranges higher and lower than Tg (ca. 60 °C), in marked contrast with the result shown by Gilding and Reed for poly(glycolide) containing a significant amount of amorphous chains.25 Our result supports the fact that the crystalline residues contained a negligibly small amount of amorphous chains.13-15 DSC. Figure 5 illustrates the DSC thermograms of the PLLA crystalline residues before the hydrolysis and after the hydrolysis for 256 days at 50 °C, 64 days at 70 °C, and 8 days at 97 °C. A melting endothermic peak can be observed at 150-190 °C for all the hydrolysis temperatures throughout the hydrolysis periods studied here, whereas no glass transition endothermic peak at around 60 °C was seen throughout the hydrolysis periods. This supports that the crystalline residues contained a trace amount of amorphous chains as revealed by X-ray diffractometry in our previous

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0.4 °C, were observed at 64 days (50 °C), 16 days (70 °C), and 2 days (97 °C), respectively. These initial increases in Tm were followed by rapid and monotonic decreases to 164.5 °C at 256 days (50 °C), to 172.2 °C at 64 days (70 °C), and to 171.4 °C at 8 days (97 °C). Such an initial increase of Tm has been ascribed to a decreased lattice disorder during the hydrolysis,15 whereas the latter decrease of Tm has been ascribed to a decreased crystalline thickness or an increased surface free energy per unit weight. No significant decrease of Tm was observed at 37 °C even after the hydrolysis for 512 days. The large deviation of the observed Xc values from 100% despite the trace amount of amorphous chains may be due to a high surface free energy of the crystalline residues whose surface was neighboring the air (or nitrogen during DSC scanning) compared with that of the crystalline regions in PLLA materials before hydrolysis, in which the crystalline regions are neighboring the same kind of chains in an amorphous state. This effect increases with decreasing the molecular weight or the thickness of the crystalline residues. Melting Temperature and “Fold Surface” Free Energy. To investigate the relationship between Tm and Mn of the PLLA crystalline residues, Tm is plotted in Figure 7a as a function of Mn. Evidently, Tm decreased monotonically with decreasing the Mn. According to the procedure reported in our previous articles,12,26,27 Mn was converted to crystalline thickness (lc) using the following eq 2: lc (nm) ) 0.288(0.80Mn)/72.1

Figure 6. Tm (a) and Xc (b) of PLLA crystalline residues (extendedchain crystallites) hydrolyzed at different temperatures as a function of hydrolysis time.

studies.14,15 The melting peaks shifted to a lower temperature, irrespective of the hydrolysis temperature. Despite the negligibly small amount of the crystallizable amorphous chains, a crystallization exothermic peak at around 80 °C was observed at 97 °C in 2 days in contrast with the results for the temperature range below 70 °C, and this peak became large with hydrolysis time. Corresponding with the crystallization peak, a melting subpeak at around 160 °C appeared at 97 °C in 2 days and its peak area increased with crystallization peak area or hydrolysis time (data not shown here). This suggests that this melting subpeak and this crystallization peak were closely related with each other, and, therefore, the melting subpeak is attributable to the crystallites formed or recrystallized at around 80 °C during DSC heating. However, we cannot give a clear mechanism for the crystallites formation or recrystallization during DSC heating. The Tm and Xc of the PLLA crystalline residues hydrolyzed at different temperatures are plotted in Figure 6 as a function of hydrolysis time. Very small increases in Tm, 0.4, 1.1, and

(2)

where the chains in the crystalline residues are assumed to be a 103 helix with a length of 28.8 nm along the c axis28,29 and amorphous chains on the “fold surface” are negligibly short compared with the crystalline chains, while 0.80 is the parameter that converts Mn for the polystyrene standard to absolute Mn on the basis of the result in a previous article14 and 72.1 is the mass per mole of half a lactide unit. Tm is plotted in Figure 7b as a function of lc-1 according to the Thomson-Gibbs equation [eq 3]:30 Tm (K) ) Tm0[1 - 2σe/lcFc∆H0]

(3)

where Tm0, σe, ∆H0, and Fc are the equilibrium Tm, specific fold surface free energy, heat of fusion per unit mass, and crystal density, respectively. It should be noted that in this study σe is not for the “fold surface” but for the surface composed of very short chains with a free end. As seen in Figure 7b, eq 3 gives an appropriate expression for the crystalline residues having the surface composed of very short chains with a free end for Tm and Mn exceeding 170 °C and 4000 g mol-1, respectively. The value at 50 °C and 256 days (Mn ) 2.74 × 103 g mol-1, Tm ) 164.9 °C), which is not shown in Figure 7b, gave a large deviation from the line for the periods below 192 days. Though the reason for it is unclear to us, it seems to occur when the molecular weight or Tm decreases below a critical value. On the other hand, the long period (L) and lc of the PLLA films crystallized to have no free amorphous region outside the spherulites at different temperatures from the melt using small-angle X-ray diffractometry in our previous study.14 The lc value can be calculated using eq 4 from L, ∆Hm, and ∆Hm-

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Figure 8. Tm of crystallized PLLA films crystallized at different temperatures as a function of (L∆Hm)-1.

Figure 7. Tm of PLLA crystalline residues (extended-chain crystallites) hydrolyzed at different temperatures as functions of Mn (a) and lc-1 (b).

(100%) values: lc (nm) ) L∆Hm/∆Hm(100%)

(4)

Substitution in eq 3 under the assumption that ∆H0 equals ∆Hm(100%) yields Tm (K) ) Tm0[1 - 2σ/LFc∆Hm]

(5)

According to eq 5 and using the Tm, L, and ∆Hm values in the article,14 Tm is plotted in Figure 8 as a function of (L∆Hm)-1. The Tm0 values of the crystalline residues (extended-chain crystallites) obtained by extrapolation of three lines of Tm to lc-1 ) 0 (Figure 7b) and that of the crystallized PLLA films evaluated by extrapolation of the line of Tm to (L∆Hm)-1 ) 0 (Figure 8) are shown in Table 2, together with reported Tm0 values.16,31-35 The Tm0 values of the crystalline residues, 464.5 K (191.3 °C), 464.9 K (191.8 °C), and 464.5 K (191.3 °C), for 50, 70, and 97 °C, respectively,

are significantly lower than other values shown in Table 2. However, the evaluated Tm0 values are comparable with those reported for the PLLA films hydrolyzed in PBS (471 K, 198 °C)26 and in the presence of proteinase K (472 K, 199 °C)27 using eq 3. In the refs 26 and 27, the crystallized PLLA films were hydrolyzed to a great extent, but the surfaces of the crystalline residues after the hydrolysis were composed of not only the very short chains with a free end but also a significant amount of folding chains. In other words, the crystalline residues in the refs 26 and 27 cannot be regarded as extended-chain crystallites. From the slope values of three lines of Tm in Figures 7b and 8, the σe values were calculated using eqs 3 and 5, respectively. Table 2 summarizes the σe values obtained using 1.29 g cm-3 (ref 18) for Fc, together with reported values.17,19,31,32,36 The σe values are given for ∆Hm(100%) ) 135 and 93 J g-1, which are abbreviated as σe(135) and σe(93), respectively. The σe(135) and σe(93) values estimated for the crystalline residues (extended-chain crystallites) having the surface composed of very short chains with a free end were 55.6-56.4 and 38.3-38.8 erg cm-2, respectively. The σe(135) and σe(93) values estimated from Figure 8 using eq 5 are identical (39.9 erg cm-2) because ∆Hm(100%) is not explicitly contained in eq 5. In marked contrast with Tm0 values, the σe values vary dramatically depending on the procedure and the parameters used for their calculation. Therefore, we discuss only the difference between the σe values obtained by the same procedure and the parameters using the Thomson-Gibbs equation in the present article. The σe values for the crystalline residues (extended-chain crystallites) having the “fold surface” composed only of very short chains with a free end, 55.6-56.4 erg cm-2 for ∆Hm(100%) ) 135 J g-1, are significantly higher than that for the crystalline regions having the “fold surface” composed of three types of chains (folding chains, tie chains, and the chains with a free end; 39.9 erg cm-2). In other words, the surface structural change from the latter (three types of chains) to the former (very short chains with a free end)

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Table 2. Equilibrium Melting Temperature (Tm0) and “Fold Surface” Free Energy (σe) for PLLA Having Different “Fold Surface” Structures

Tm0(TG)a (K)

Tm0(HW)b (K)

Tm0(M)c (K)

σe(135)d (erg cm-2)

σe(93)e (erg cm-2)

Crystalline Residues (Extended-Chain Crystallites): Very Short Chains with a Free End (Neighboring the Air) hydrolyzed at 50 °C 464.5 55.8 38.4 hydrolyzed at 70 °C 464.9 55.6 38.3 hydrolyzed at 97 °C 464.5 56.4 38.8 Kalb and Pennings31 Vasanthakumari and Pennings32 Tsuji and Ikada16,33,34 Huang et al.19 Miyata and Masuko17 Abe et al.35 Tsuji and Ikarashi14

Crystallized Films: Folding Chains, Tie Chains, Chains with a Free End 488 480 478-485

61 60

63-107 500.1 478.3f

39.9f

39.9f

Single Crystals: Folding Chains, Trace Amount of Chains with a Free End (Neighboring the Air) 53 ( 4 75

Hoffman et al.36 Kalb and Pennings31

a The T 0 value obtained by the Thomson-Gibbs equation.30 b The T 0 value obtained by the Hoffman-Weeks procedure.37 c The T 0 value obtained m m m by the Marand et al. procedure.38,39 d The σ value for ∆Hm(100%) ) 135 J g-1. e The σ value for ∆Hm(100%) ) 93 J g-1. f The given values have been calculated in the present study on the basis of the data reported in ref 14.

during hydrolysis of crystallized PLLA materials increases their σe values,14 which can be one of the causes for their decreased Tm by hydrolysis. As mentioned previously, probable main cause for the increased σe by hydrolysis is that the crystalline regions in PLLA materials before hydrolysis are neighboring the same kind of chains in an amorphous state, whereas the surfaces of the crystalline residues are neighboring a different kind of substance other than PLLA chains, air (or nitrogen during DSC scanning). Here, we do not discuss the σe values 38.3-38.8 erg cm-2 for ∆Hm(100%) ) 93 J g-1 because 93 J g-1 is known to be smaller than the real ∆Hm(100%). A lower Tm appeared in addition to the main Tm when crystallized PLLA films were hydrolyzed in the presence of proteinase K to a great extent.27 In such enzymatic hydrolysis, the tie chains and the chains with a free end in the amorphous regions were predominantly hydrolyzed and removed but the folding chains were not hydrolyzed, resulting in formation of the surface composed of very short chains with a free end and folding chains, which are neighboring the air. The surface structure and environment of the crystalline regions after the enzymatic hydrolysis approach those of the crystalline residues in this study because both surfaces have very short chains with a free end neighboring the air. This finding and eq 3 strongly suggest that the σe value for the surface composed of very short chains with a free end in the crystalline residues (extended-chain crystallites) is much higher than that for the initial “fold surface” composed of the three types of chains before hydrolysis. This agrees well with and supports the previously mentioned result. The gradual decrease in Tm at the late stage of hydrolysis of crystallized PLLA materials in vitro in PBS or Ringer solution and in vivo12 is attributable not only to a decrease in crystalline thickness but also to structural and environmental changes at the “fold surface”. Further studies and accumulation of data are required for the detailed comparison between the σe value for the surface composed of very short chains with a free end and those for the surfaces having different structures.

Conclusions The following conclusions can be derived from the experimental results for the PLLA crystalline residues (extended-chain crystallites) hydrolyzed in PBS at different temperatures. (1) The hydrolysis of the crystalline residues proceeded from their surface composed of very short chains with a free end along the chain direction irrespective of hydrolysis temperature, but the hydrolysis from their lateral surface could not be traced. (2) The Mn of the crystalline residues decreased monotonically and linearly with hydrolysis time, and the average hydrolysis rates evaluated from the Mn changes were 28, 86, and 710 g mol-1 day-1 at 50, 70, and 97 °C, respectively. (3) The activation energy of hydrolysis for the crystalline residues (extended-chain crystallites) was evaluated to be 18.0 kcal mol-1 (75.2 kJ mol-1), which is significantly higher than 12.2 kcal mol-1 obtained for PLLA in the melt in the temperature range of 180-250 °C. (4) Significant and monotonic Tm and Xc decreases were observed with hydrolysis time at 50, 70, and 97 °C after their initial very small increases, excluding the monotonic Xc decrease at 97 °C with no initial increase. The Tm decrease of the PLLA crystalline residues reflects solely their decreased thickness, whereas the Tm decrease of the crystallized PLLA materials reflects not only the decreased thickness of the crystalline regions but also the structural and environmental changes at their “fold surface”. (5) The Tm0 of the crystalline residues (extended-chain crystallites) was estimated to be 464.5-464.9 K (191.3191.8 °C). The σe values for the surface composed of very short chains with a free end, which are neighboring the air (or nitrogen during DSC scanning), were calculated to be 55.6-56.4 erg cm-2 for heat of fusion per unit mass ) 135 J g-1. The obtained σe values are significantly higher than that for the surface composed of folding chains, tie chains, and chains with a free end, which are neighboring the same kind of amorphous chains (39.9 erg cm-2).

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