Effects of Physical Aging, Crystallinity, and Orientation on the Enzymatic Degradation of Poly(Lactic acid)

H U A CAI,' VlPUL DAVE,' RICHARD A. CROSS;

and STEPHEN P. McCARTHY','

Departments of 'Plastics Engineering and 'Chemistry, NSF Center for Biodegradable Polymer Research, University of Massachusetts Lowell, Lowell, Massachusetts 01854

SYNOPSIS

The effects of physical aging, degree of crystallinity, and orientation of poly(1actic acid) (PLA) were studied using differential scanning calorimetry (DSC) and wide angle X-ray The samples of PLA with 96% [ L ]and 4% [ D ]contents were prepared scattering (WAXS). by injection molding. The physical aging of PLA strongly depended on time and temperature. The change of rate of physical aging was very fast initially and slowed down as time increased. The enzymatic degradation of PLA was carried out with proteinase K at 37C at a pH value of 8.6 in a Tris/HCl buffer solution. The enzymatic degradation rate was found to decrease as a function of physical aging (i.e., excess enthalpy relaxation). The rate of enzymatic degradation of PLA decreased with the increase in crystallinity. A threshold was observed when the heat of fusion was less than 20 J/g. The weight loss of PLA with a low level of crystallinity had no apparent change during any period of testing time. The rates of enzymatic degradation of stretched and injection-molded specimens were comparable.
0 1996 John Wiley & Sons, Inc.

Keywords: poly(1actic acid) biodegradation crystallinity orientation physical aging

INTRODUCTION
Poly(1actic acid) or polylactide polymers have attracted attention in recent years due to their biocompatibility with biological tissues,'*2degradability i n vitro and in ~ i u o , ~ and - ~ good mechanical prope r t i e ~ . Extensive ~.~ work has been carried out by several investigators in understanding the morphological properties of poly(L-lactic acid) (PLLA). In particular, considerable progress has been made in elucidating the crystalline structures-10and crystallization of PLLA. Recently, detailed studies were carried out to investigate the influence of physical aging on the viscoelastic behavior of PLLA,15 and the effects of water sorption on the internal motions in PLLA and other related polyrners.l6 The influence of morphology (crystalline and amorphous) on the degradation of PLLA was con* To whom correspondence should be addressed.
Journal of Polymer Science: Part B: Polymer Physics, Vol. 34,2701-2708 (1996) 0 1996 John Wiley & Sons, Inc. CCC 0887-6266/96/162701-08

ducted in aqueous media for periods up to 2 years.17 It was determined from this study that the highly crystalline residues appear to be very resistant to degradation, and that degradation proceeds more rapidly in the center than at the surface for both the crystalline and the amorphous specimens. Currently, there is an increasing interest in utilizing PLA for disposable and biodegradable plastic products." Dramatic effects of polymer stereochemistry and the resulting crystalline morphology on enzymatic degradation of poly(P-hydroxybutyrate)" and PLAZ0 have been studied extensively in our laboratory. The importance of morphology on degradation has been established for PLA, and a more detailed treatise on this subject is needed as the information generated will have a great impact in interpreting the degradation behavior of PLA prepared from different processing techniques for various applications. In the present study, we report the effects of physical aging, crystallinity, and orientation of PLA on enzymatic degradation of PLA (96% [L]).
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Sample Preparation

t
w

L
27 hours
144 hours

Injection-Molded Samples

s
0

a
u1

PLA pellets were dried in a vacuum oven at 40C for 8 h. The pellets were injection molded using a 20 Ton Negri-Bossi injection molding machine with a single cavity, fan-gated mold which was specifically designed to produce thin injection-molded specimens with 75 X 12.7 X 0.5 mm dimensions. Injection molding was carried out at a melt temperature of 195"C, injection and packing time of 10 s, and injection pressure of about 70 MPa.
Aged Samples

one month

t
60
80

I00

Temperature,' C

Figure 1. DSC curves of PLA aged at room temperature

for various time.

EXPERIMENTAL
Materials

The physical aging of the resulting injection-molded samples was studied with a differential scanning calorimeter. Each sample was heated to 180C a t a heating rate of 10"C/min and was then rapidly quenched to about -40C in order to erase any previous thermal history. In order to study the effect of time on physical aging, the quenched samples were stored at ambient temperature (18-20C) for various periods of time. In order to study the effect of temperature on physical aging, the quenched samples were kept at the selected temperature for a fixed time period of 24 h.
Annealed Samples

PLA (96% [L]) was supplied by Cargill Inc. in pellet form. The number-average molecular weight was 137,000 as determined by gel permeation chromatography using polystyrene standards.
4

Different crystallinities of the PLA samples were obtained by isothermal crystallization of the injection-molded specimens in an oven at 105C for various periods of time. The dimensional changes were

4 ,

0 3 . 0
3.5
4.0
5

log (Ta. min)

30

60

90

120

150

Aging time, (hours)

Figure 2.

Excess enthalpy relaxation as a function of aging time for PLA.

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Aging temperature

min for 4 min. After stretching, the samples were quenched in a cooling cylinder filled with dry ice. The amount of orientation was qualitatively determined by measuring the unrestrained linear shrinkage (%) [ASTM D 27321 of the oriented sample after annealing them in an oven at 70C for 1 h. The shrinkage was determined by the ratio of the change in length to the original length of the samples.
Thermal Analysis

1
40
60 80 100

20

Temperature CC)

Figure 3 . DSC curves of PLA annealed for 24 h at different temperature.

The glass transition temperature ( Tg), melting temperature (T,,,), and heat of fusion (AH,,,) were measured by using a DuPont DSC 912 differential scanning calorimeter equipped with a TA 9000 data station with samples ranging in weight between 4 and 10 mg and at a heating rate of 10"C/min under a nitrogen purge. The T,,, and AH,,, were determined from the endothermic melting peaks. The excess enthalpy of relaxation (AHEx)caused by physical aging was characterized by integrating the area under the endothermic peak which occurred at the Te
X-Ray Analysis

measured for both the aged and annealed samples and were found to be negligible.

Oriented Samples
The injection-molded PLA samples were oriented by stretching them in a tensile testing machine equipped with a temperature-controlled chamber. Stretching was performed at 60C (a, = 14 MPA) and 90C (a, = 8.7 MPA) at a strain rate of 0.5"/
5

Wide angle X-ray measurements were conducted for the oriented samples on a Rigaku Geigerflex camera operating at a voltage of 40 kV and a current of 25 mA. Nickel-filtered CuKa radiation (A = 1.542 A) was used.
Scanning Electron Microscopy (SEM)

Electron micrographs were obtained on samples which were collected before and after the enzymatic

20

40

60

80

100

Temperature of aging, (C)

Figure 4 . Excess enthalpy relaxation as a function of aging temperature for PLA.

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100

200

300

Annealing time (min)

Figure 5 . Heat of fusion of PLA as a function of annealing time at 105C.

degradation tests by using an scanning electron microscope model ISI-IC130. The specimens were withdrawn after 66 h. These specimens were mounted on stubs and were carbon coated using a sputter coater.

Density Measurement

A density gradient column was prepared in accordance with ASTM D1505. All the measurements were carried out at 23C. Distilled water and aqueous sodium bromide (NaBr) solutions were used as the liquid system to prepare the column. The column was prepared to cover the density range from 1.0 to 1.4 g/cc.

Enzymatic Degradation Study

I 6

12

18

24

28

Figure 6. Wide angle X-ray scattering of PLA samples: (a) injection molded, (b) stretched at 90C, (c) stretched at 60C.

Weight loss studies on the PLA samples were carried out with a modification to the existing literature method.*' PLA samples (10 X 10 mm) with an approximate thickness of 0.5 mm were placed into vials containing 5 mL of Tris/HCl (pH = 8.6), 1.0 mg proteinase K (Sigma, lyophilized powder, 15 unit/ solid), and 1.0 mg of purified sodium azide (Sigma). The water used to prepare the above solution was purified by a Barnstead NAN0 pure water purification system. For a given experiment, three replicate samples in separate vials were used for weight loss measurement at a specified incubation time. The sample/enzyme incubations were carried out at 37C in a rotary shaker (200 rpm). The buffer/enzyme system solution was changed every 24 h to restore the original level of enzyme activity. The samples were periodically removed, washed with distill water, and then dried in vacuum (0.005 mg) for 36 h at room temperature. The standard deviations for all the weight loss values were within 5%. Controls were also run for all the samples under similar conditions

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200

*
150
C U -

1 month

E E
W

VI

100

._
c m

'

50

100%

0 0
20
40 Time 01 degradation (hours)

322.48 ug 80

60

Figure 7.

Normalized weight loss of PLA as a function of aging time.

without enzyme but there was no weight loss observed up to the total experiment time of 66 h.

RESULTS AND DISCUSSION

Physical Aging of PLA

t----1
5Pm
Figure 8. Scanning electron micrographs of degraded samples after 66 h: (a) aged, (b) deaged.

DSC was used to determine the physical aging behavior of PLA. All samples were quenched from the melt to -40C in order to erase any previous thermal history. Figure 1 shows the DSC thermograms of PLA aged a t room temperature for different periods of time. It is observed that the endothermic peak a t the Tg increases with an increase in aging time as a result of an increase in the excess enthalpy of relaxation. The extent of physical aging was obtained from the area under the endothermic peak at the glass t r a n ~ i t i o n . ' Figure ~ ? ~ ~ 2 shows the relationship between the excess enthalpy relaxation, AHEX, and aging time (t,) for PLA. The rate of physical aging is very fast initially and decreases as time increases. The value of AHEx increases from 3.79 to 4.91 J/g as the aging time increases from 144 h to 1 month. It is not known whether an equilibrium state is achieved after an aging time of 1 month. The data of A H E X are replotted in the inset of Figure 2 as a log-log plot. This apparent linearity indicates an exponential relationship.22 The mobility of the polymer chains is directly related to temperature as it affects the ability to eliminate excess free volume. Figure 3 shows the effect of temperature on the physical aging of PLA for 24 h. In this study, all of the samples had similar thermal history and were aged a t different temperatures. The AHExvalue increases at 25C and 37C.

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300

10

20

30

40

50

Heat of fusion (J/g)

Figure 9. Normalized weight loss as a function of heat of fusion after 85 h of degradation time.

This is a result of the rate-controlling effect of temperature on the aging process. It is clearly observed that the MEx value reached a maximum value when the sample was aged at 37C. When the aging temperature was increased to the glass transition temperature (60"C), the excess enthalpy relaxation was greatly reduced. Physical aging was no longer observed when the aging temperature progressed above the Tr A slight increase in the Tgvalue (- 3C) was observed for samples aged at 50C. This is expected due to the reduction in free volume and the consequent reduction in the molecular mobility during aging. Figure 4 demonstrates the relationship between excess enthalpy relaxation as a function of

aging temperature. The maximum physical aging was seen to occur a t about 37C.
Crystallization of PLA

The melting point (T,)and heat of fusion ( A H m ) were measured by DSC. Figure 5 shows the heat of fusion of PLA annealed a t 105C for different lengths of time. It is observed that the heat of fusion (i.e., crystallinity) value increases with an increase in the annealing time. The crystallization rate appears to have leveled off after about 100 min as the heat of fusion has reached a plateau value of about 40 J/g.

200 Stretched at 6OoC Stretched at 90C

,.,A

E
04

150

0 -

2 100

'
E 04 .-

58

0
0
20
40

60

80

Time 01 degradation (hours)

Figure 10. Weight loss as a function of time of injection-molded samples and stretched
samples at 60C and 90C.

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Orientation of PLA

lecular orientation in the direction of flow during the injection molding process.
Enzymatic Degradation of PLA

The injection-molded samples were oriented in order to observe the influence of molecular orientation on degradation. The density values of injection-molded samples were found to be 1.2422 g/cc. The density of the oriented samples was determined to be 1.220 g/cc for 60C stretching and 1.2435 g/cc for the 90C stretching. The decrease in density for the sample oriented a t 60C is due to a Poisson's ratio below 0.5 for temperatures near the Tg. There is also evidence of crazing in the samples during the orientation process, which was carried out a t the temperature near the Tg(60C) of PLA. It should be noted that the density value of the oriented sample a t 90C is similar t o that of the unoriented sample. Figure 6(a-c) shows the WAXS patterns of injection-molded sample and the oriented samples a t 90C and 60"C, respectively. The injectionmolded samples are seen to be completely amorphous [Fig. 6(a)] as has been verified by the DSC results. The oriented sample a t 90C did not exhibit any residual orientation as the temperature of orientation was above the glass transition temperature (i.e., 60C) of PLA, which probably caused the relaxation of the polymer chains [Fig. 6(b)]. The oriented sample a t 60C displayed a sharp reflection a t 28 = 16, which represents a dspacing of 5.57 A. This indicates preferential molecular orientation of the material during the stretching process, which is retained upon cooling in the sample. The degree of orientation was qualitatively determined by the unrestrained linear thermal shrinkage experiment. The change in length of the samples was measured after annealing them a t 70C for 1 h. The shrinkage was 27% for the unoriented injection-molded sample, 47% for the oriented sample a t 9O"C, and 70% for the oriented sample a t 60C. The results indicate that the orientation is the highest a t 60C as the shrinkage value is the maximum for that particular sample. The shrinkage was caused by the relaxation of the residual strains in the molecules during the annealing process a t a temperature (i.e., 70C) above the Tg of PLA. This observation also confirms the X-ray observations. However, the sample oriented a t 90C does not show any preferential orientation in the X-ray pattern but does exhibit 47% linear shrinkage. This indicates that the sample is indeed oriented to some extent, which is, however, not detected by the X-ray. The unoriented sample also demonstrates a 27% shrinkage caused by the mo-

Physical Aging Effects

Figure 7 shows the normalized weight loss of PLA as a function of time during enzymatic degradation of various aged samples. The weight loss of the quenched, deaged samples was much higher than the aged samples (24 h and 1 month) after degradation for 66 h. This indicates that the decrease in the mobility of the polymer chains upon aging and the reduction in free volume hinder the enzymatic attack on the polymer chains compared to that of the deaged samples. It is interesting to note that all three samples have almost the same weight loss after only 8 h of degradation, which indicates that this phenomenon is not a surface effect, even though the degraded surface morphology was quite different. It should be noted that maximum physical aging occurred at 37"C, which is the temperature a t which the enzymatic degradation study was carried out. Therefore, the samples are being physically aged during the degradation test. Figure 8(a,b) shows the scanning electron micrographs of the surfaces of the aged and deaged samples after enzymatic degradation for 66 h. Aging may play a role in altering the accessibility of the enzyme to attack the polymer chains, which results in a different surface morphology of the aged and deaged sample.
Crystallinity Effects

Figure 9 shows the relationship between enzymatic degradation of PLA as a function of heat of fusion (crystallinity). The weight loss values are almost comparable below the heat of fusion value of 20 J/ g. This is possibly due to the fact that the amorphous regions are easily accessible for enzymatic attack as this limited amount of crystallinity does not inhibit degradation. However, there is a sudden drop observed in the weight loss values beyond the heat of fusion of 20 J/g, which indicates that above a critical amount of crystallinity degradation is inhibited.
Orientation Effects

Figure 10 compares the weight loss in injectionmolded and oriented PLA samples. The degradation tests were carried out immediately after the orientation experiment in order to avoid physical aging effects. It is observed that the rate of enzymatic deg-

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radation of PLA is the highest for the unoriented sample followed by the samples oriented at 60C and 9O"C, respectively. Due to the molecular orientation of sample stretched at 60"C, its degradation is lower than the unoriented sample. However, the surface area of the oriented sample increased due to the presence of voids (as confirmed by density) which were formed during the stretching experiments. Therefore, the degradation is expected to be higher for these materials as the increase in the surface area makes them more susceptible to enzymatic attack. The degradation of the sample oriented at 90C is the lowest due to some degree of preferential orientation and the absence of void formation during the stretching experiment.

CONCLUSION S
The effects of physical aging of PLA have been found to depend on both aging time and temperature. A maximum value of excess enthalpy was observed a t 37C. It was determined that the deaged samples had a much higher weight loss than the aged samples. It was possible to vary the crystallinity of PLA by annealing the samples at different temperatures. Enzymatic degradation was relatively high for materials with low crystallinity, and dropped dramatically after the samples had a critical amount of crystallinity which corresponds to the heat of fusion value of 30 J/g. Injection-molded PLA samples were successfully oriented at 60C and 90C as was confirmed by the WAXS patterns and the unrestrained linear shrinkage experiment. However, it seems that the void formation (crazing) during the stretching process a t 60C resulted in almost similar degradation rates for the oriented samples once the increase in surface area was accounted for. The degradation of the samples oriented at 90C is lower than the unoriented, injection-molded samples.

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R E F E R E N C E S AND NOTES
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Received July 1 1 , 1995 Revised May 2, 1996 Accepted May 22, 1996