Morphological investigation on melt crystallized polylactide homo- and stereocopolymers by enzymatic degradation with proteinase K

Morphological Investigation on Melt CrystallizedPolylactide Homo- and Stereocopolymers by EnzymaticDegradation with Proteinase K

YONG HE,1,2 TONG WU,1 JIA WEI,1 ZHONGYONG FAN,1 SUMING LI1,2

1Department of Materials Science, Fudan University, Shanghai 200433, People’s Republic of China

2Max Mousseron Institute on Biomolecules, UMR CNRS 5247, Faculty of Pharmacy,University Montpellier I, 34060 Montpellier, France

Received 14 September 2007; revised 11 December 2007; accepted 10 February 2008DOI: 10.1002/polb.21430Published online in Wiley InterScience (www.interscience.wiley.com).

ABSTRACT: Polylactide (PLA) homo- and stereocopolymers containing 100, 98, 96, 94,and 92% L-lactyl units, respectively, were synthesized by ring opening polymerizationof L-lactide and DL-Lactide, using zinc lactate as catalyst. Differential scanning calori-metric analysis measurements show that incorporation of D-lactyl units leads todecrease of the crystallization rate of the copolymers. However, the crystallizationmechanism and the amount of crystallizable fraction are not affected. The enzymaticdegradation was performed at 37 8C in a pH 8.6 Tris buffer containing proteinase K.Two distinct morphologies were obtained by melt crystallization for PLA films withca. 80 lm of thickness. It is confirmed that proteinase K can degrade both the freeand confined amorphous regions. Lamella stacks in spherulites retain their orienta-tion during enzymatic degradation. PLA crystal morphologies are affected by the con-tent of D-lactyl units. Factors such as the nucleus location and the D-lactyl units’exclusion as amorphous fraction were considered to elucidate the observed PLAspherulite morphologies. Infrared spectroscopy and mass loss measurements werealso combined to better understand the degradation behaviors. VVC 2008 Wiley Periodicals,

Inc. J Polym Sci Part B: Polym Phys 46: 959–970, 2008

Keywords: crystallization; degradation; morphology

INTRODUCTION

In the family of aliphatic polyesters, poly(L-lac-tide) (PLLA) and related copolymers have beenattracting more and more attention mainly dueto their biodegradability, biocompatibility, andgood mechanical properties. These biodegradablepolymers present great interest for medical andenvironmental applications such as drug deliv-

ery systems, sutures, surgical implants, andcommodity resins.1–5

In 1981, Williams first reported that hydroly-sis of polylactide (PLA) is catalyzed by protein-ase K from Tritirachium album.6 Since then,the enzymatic degradation of PLA has beenreported by many research groups. The mainfactors affecting the enzymatic degradability ofPLA have been clarified. Proteinase K prefer-entially degrades L-lactyl units as opposed toD-lactyl ones, poly(D-lactide) being not degrad-able.7 Crystallinity also significantly affects thedegradation rate.7–12 The enzymatic degrada-tion preferentially occurs in the amorphousregion of semicrystalline PLA polymers.10 Bulk

Correspondence to: Z. Fan or S. Li (E-mail: [email protected] or [email protected])

Journal of Polymer Science: Part B: Polymer Physics, Vol. 46, 959–970 (2008)VVC 2008 Wiley Periodicals, Inc.

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hydrophilicity appears to be another importantfactor since water uptake can lead to swellingof polymers and thus facilitate enzymatic deg-radation.11

Since the pioneer work by Fischer et al.,13 thecrystallization behavior of PLA homo- and ste-reocopolymers has been extensively studied.14,15

Crystallinity and spherulite growth rate sub-stantially decrease with increasing D-lactyl unitsin the copolymers. Both the rejection of D-lactylunits from PLA crystals and inclusion in thecrystals were suggested in the crystallizationprocess.13,14 On the other hand, the morphologyof PLA has also been investigated. Lamella sin-gle crystals of PLA were obtained by variousmeans, with morphologies of hexagonal, trun-cated-lozenge, and lozenge-shapes.13,16–18 Fisheret al. reported a selective degradation of thespherulite centers when alkaline degradationwas applied to melt crystallized PLA.13 Tsuji et al.suggested that alkaline degradation of PLLAwould cause lamella stacks lose their orientationin spherulites by using polarized optical micros-copy (POM).19 In contrast, enzymatic degrada-tion of PLLA single crystals has been shown toprogress from the edges of lamella.18 However,the morphologies of melt crystallized PLA homo-and stereocopolymers have not been investi-gated in detail by means of enzymatic degrada-tion in the presence of proteinase K on spheru-lite scale.

In a series of papers, we reported on the en-zymatic degradation of PLA in the presence ofproteinase K.10,11 Comparison between variousPLA homo- and stereocopolymers confirmed thatproteinase K preferentially degrades L-lactylunits as opposed to D-lactyl ones. Blends ofPLLA with poly(e-caprolactone) (PCL) have alsobeen considered. The selective enzymatic degra-dation in the presence of pseudomonas lipaseor proteinase K revealed the phase-separatedstructures of the blends by using environmentalscanning electron microscope (ESEM).20 Re-cently, we studied the crystallization behaviorand crystal morphology of PLLA by using POMand differential scanning calorimetric analysis(DSC).21,22 In this work, a series of PLA homo-and stereocopolymers were synthesized by usingzinc lactate as catalyst. Melt crystallized PLAfilms were allowed to degrade in presence of pro-teinase K. The morphologies of degraded filmswere examined by using ESEM, together withDSC and Fourier transform infrared spectropho-tometer (FTIR).

EXPERIMENTAL

Materials

L-lactide and DL-lactide were purchased fromPurac (Netherlands). Zinc lactate, Trizma base,and sodium azide were obtained from Merck.Trizma/HCl and proteinase K (30 U/mg) in theform of lyophilized powder were supplied bySigma.

Methods

PLA homo- and stereocopolymers, namelyPLA100, PLA98, PLA96, PLA94, and PLA92 weresynthesized by ring-opening polymerization of100/0, 96/4, 92/8, 88/12, and 84/16 L-/DL-lactidemixtures. The subscripts of the samples’ namesare the theoretical values of L-lactyl contentaccording to the feed ratio. Zinc lactate (0.05%by weight) was used as catalyst. Polymerizationwas allowed to proceed at 140 8C for 1 week.The resulting polymers were purified by the dis-solution/precipitation method using dichlorome-thane as solvent and ethanol as nonsolvent.

Enzymatic Degradation

Films of ca. 80-lm thickness were prepared bymelt compression between two glass plates, fol-lowed by isothermal crystallization at varioustemperatures for 2 h. Typically, each film wasplaced in a vial filled with 10 mL of pH 8.6 Trisbuffer (0.05 M) containing 2.0 mg of proteinaseK and 2.0 mg of sodium azide. The vials wereplaced in an oven thermostated at 37 8C. Degra-dation was allowed to proceed up to 72 h. Thedegraded films were recovered after preset deg-radation periods, washed with distilled waterand examined by using ESEM. They were thenvacuum-dried up at room temperature to con-stant weight for DSC and FTIR analysis.

Measurements

Size-exclusion chromatography (SEC) measure-ments were performed with a Waters apparatusequipped with a refractive index detector. Chlo-roform was used as the mobile phase at a flowrate of 1.0 mL/min. 20 lL samples of 0.5% (w/v)solution were injected for each analysis. The col-umns were calibrated with polystyrene stand-ards (Polysciences). Specific rotation values ofPLA were measured in chloroform at a concen-

960 HE ET AL.

Journal of Polymer Science: Part B: Polymer PhysicsDOI 10.1002/polb

tration of 8.6 g/L by using a WZG-2S polarime-ter. DSC analysis was performed with a Perkin–Elmer instrument DSC 6 calibrated withindium, the heating rate being 10 8C/min. Mor-phologies of the films were examined by using aPhilips XL30 ESEM under reduced pressurebelow 1 Torr. FTIR spectra of PLA were recordedon a Perkin–Elmer spectrum 100 at a 2 cm�1

resolution, with the sample dispersed in KBrpowder.

RESULTS AND DISCUSSION

Basic Characteristics of PLA Homo- andStereocopolymers

PLA polymers were synthesized by ring-openingpolymerization of appropriate monomer feeds.Zinc lactate is preferred to commonly used stan-nous octoate as catalyst since the former is lesscytotoxic and does not lead to chain end modifi-cations.23,24 The thus obtained PLA homo- andstereocopolymers were characterized by SEC,polarimetry and DSC. Table 1 lists the molecu-lar weights, specific rotation values, and ther-mal properties. The Mn of the polymers rangesfrom 57,000 to 143,000 with a rather narrowpolydispersity (Mw/Mn �1.9). The specific rota-tion value ([a]D20) for PLA100 was found to be�152.1o, which is in the range of values from�149o to �156o as reported in the literature.15,25

The L-lactyl contents of PLA stereocopolymersPLA98, PLA96, PLA94, and PLA92 were evaluatedfrom the specific rotation values when comparedwith that of PLA100. The calculated values are

equal to the theoretical data shown as subscriptof samples’ name (Table 1).

DSC analysis was first performed on originalpolymer samples. A second heating was thenperformed on amorphous samples obtained byquenching from melt. All the values of meltingtemperature (Tm), melting enthalpy (DHm), andmelting entropy (DSm) were included in Table 1.No melting was detected for PLA94 and PLA92

at the second heating due to the low crystalliza-tion ability. In fact, it has been reported that acontent of L-lactyl units over ca. 80% is requiredfor crystallization.26,27 PLA100 exhibits the high-est Tm, DHm, and DSm values when comparedwith PLA stereocopolymers because of its higherchain regularity. Tm of PLA100 remains thesame, whereas DHm and DSm are higher inthe second heating than in the first one. Theincrease of DHm and DSm could be related to thehigh crystallization ability of PLA100 whichcould better crystallize at a heating rate of10 8C/min than during precipitation from the so-lution. In contrast, Tm, DHm and DSm values arelower in the second heating than in the first onefor PLA98 and PLA96 samples, which seems toindicate that crystallization at a heating rate of10 8C/min contributes to less perfect lamellaethan during precipitation from the solutionbecause of lower crystallization ability.

On the other hand, glass-transition tempera-tures (Tg) were detected as 60 (61) 8C at thefirst heating run and 58 (61) 8C at the secondone. Tg is slightly higher for the first runbecause polymer chains are more confined bycrystalline domains. A slight Tg decrease is alsodetected with increasing the content of D-lactylunits.

Table 1. Molecular Weights, Specific Rotation Analysis, and Thermal Properties of PLA

Samples

Molecular Weight Specific Rotation Analysis Thermal Properties

Mn Mw/Mn [a]D20 (deg) XL (mol %)a Tm (8C) DHm (J/g) DSm (mJ�g�1�K�1)

PLA100 145,000 1.6 �152.1 100 173.7b/173.3c 22.8b/34.1c 51.0b/76.4c

PLA98 83,000 1.9 �145.8 97.9 162.7/160.7 20.9/19.2 47.9/44.2PLA96 76,000 1.5 �140.7 96.3 152.7/150.0 16.7/<0.1 39.2/<0.2PLA94 57,000 1.7 �132.0 93.4 146.0/n.d.d 16.1/n.d. 38.4/n.d.PLA92 79,700 1.6 �126.2 91.5 133.7/n.d. 12.5/n.d. 30.7/n.d.

a The L-lactyl unit fraction of these polymers (XL) was evaluated with the equation: XL

100 ¼ ½a�D20þ½a�D20ðPLA100Þ2�½a�D20ðPLA100Þ

.

b The values in the left columns are the thermal properties from DSC analysis with original samples after dissolution/precip-itation process.

c The values in the right columns are from the amorphous samples quenching from the melt.d n.d.: not detected.

ENZYMATIC DEGRADATION WITH PROTEINASE K 961


Melt Crystallization Analysis by DSC

PLA melt crystallization was analyzed by DSC.Crystallization isotherms of PLA100, PLA98 andPLA96 are presented in Figure 1. Heat flow dur-ing isothermal crystallization is strongly affectedby both the crystallization temperature (Tc) andL-lactyl content of PLA. Values of crystallizationenthalpy (DHc) are listed in Table 2. It appearsthat DHc slightly increases with increasing Tc forall three polymers, which could be assigned tothe higher chain mobility at higher tempera-tures. In contrast, DHc shows little dependenceon D-lactyl unit fraction at a given temperature,suggesting that the three PLA homo- and stereo-copolymers have comparable crystallizable frac-tions. This result might imply that comparablecrystallinity could be reached after completecrystallization because of little variation ofcrystallization thermodynamics.

Crystallization isotherms could be analyzedby using Avrami equation,28

Xc ¼ 1� exp �katnað Þ ð1Þ

where Xc is the relative crystallinity, ka is theAvrami crystallization rate constant, and na isthe Avrami exponent. A curve fitting procedurewas introduced in the analysis and the resultsshown in Table 2. The values of na are equal toca. three for all three PLA samples. Consideringthe 3-dimensional growth of spherulites as shownin previous reports,21,29 heterogeneous nuclea-tion should occur before crystal growth.30 Thisconclusion will be confirmed by ESEM investiga-tion on PLA spherulite morphologies in the nextsection. The incorporation of D-lactyl units doesnot affect the crystallization mechanism.

On the other hand, the crystallization half-time, t1/2, exhibits strong dependence on the con-tent of D-lactyl units. The crystallization rate, asreflected by 1/t1/2, decreases with increasing D-lactyl content. It can be considered that D-lactylunits are included in polymer chains as defects,thus reducing the regularity of polymer chains,and affecting the diffusion kinetics and crystalli-zation kinetics. As a result, the relatively slowercrystallization rates for PLA94 and PLA92 con-tribute to the disappearance of melting points asshown in Table 1.

It should be noted that the t1/2 value isslightly lower than the peak crystallization time(tp) which could be directly obtained from thecrystallization isotherms. This discrepancy is

ascribed to the fact that the induction period ofcrystallization is not taken into account for theestimation of t1/2 value. Therefore, the value oftp should not be taken as an indication of the

Figure 1. Isothermal melt crystallization of PLA100,PLA98, and PLA96 samples at various Tc’s. The Tc’sare indicated in the figure.

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overall rate of crystallization.31 In addition, themaximum overall rate of crystallization appearsat ca. 105 8C, which is not significantly affectedby the content of D-lactyl units.

Morphology Examination by ESEM AfterEnzymatic Degradation

The films after melt crystallization were exam-ined by using ESEM. Smooth surfaces wereobserved without detection of any spherulites.After degradation in enzyme-containing Trisbuffer, the films became whitish. ESEM waspreferably used to monitor the changes of sur-face morphology of degraded films, because itdoes not necessitate high vacuum or gold coat-ing which often results in artifacts.

Crystallization at 135 8C

PLA100 Films. Figure 2 shows the ESEMimages of PLA100 film crystallized at 135 8C anddegraded by enzyme for various periods. Beforedegradation, the films appeared rather smoothwithout visible defects [Figure 2(a)], which wereoften observed in the liquid nitrogen quenchedfilms after crystallization.22 After 24 h degrada-tion, the film was slightly eroded with partialremoval of amorphous domains [Figure 2(b,c)].The boundaries between spherulites could bedistinguished and the size was estimated to beca. 400 lm. The irregular shapes of spherulitescould be attributed to the fact that the radialgrowth of spherulites is stopped by collisionwith surrounding ones.

It is of interest to note the appearance of twodifferent kinds of morphology in the central partof the spherulites Figure 2(b,c). Spherulitic fibrilswere detected along radial direction of the spher-

ulite in Figure 2(b), whereas disordered morphol-ogy composed of numerous spherulitic fibrils wasobserved in Figure 2(c). It should be noted thatproteinase K preferentially degrades amorphouspart of PLLA prior to crystalline region. Thecracks between spherulitic fibrils were formedbecause the amorphous region confined in spher-ulites was slightly degraded at the surface after24 h degradation. Those two kinds of morphologyin Figure 2(b,c) seem to result from differentnucleation locations. If the nucleation occurs onthe surface, lamella stacks grow on parallel tothe surface. Otherwise, lamella stacks growgradually to the surface from inside, whereaspart of amorphous PLA100 was entrapped in thefilm surface and the spherulite fibrils.

Furthermore, the spherulite surface becamegradually smoother when growing outwardsfrom the center. Considering the film thicknessand spherulite size, the growth direction of thelamella stacks could gradually incline to be par-allel to the surface during crystal growthalthough the nuclei are located inside. Fisher etal. have observed selective degradation of thespherulite center by etching melt crystallizedPLA with alkaline solution. The authors sug-gested that the center contains higher fractionof disordered material or defects than the outerregions of the spherulites.13

In semicrystalline polymers, the bulk is com-posed of three different domains: crystalline frac-tion, free amorphous region, and confined amor-phous region between the lamella stacks. Tsuji etal. reported that the enzymatic degradability ofconfined amorphous regions was similar to thatof free ones.28 In Figure 2(d), lamella stacks inthe growth boundary of the spherulite were dis-closed in side face as both the free and confinedamorphous regions were eroded. Lamella stacksin the side face did not collapse although the con-

Table 2. Melt Crystallization Analysis of PLA100, PLA98, and PLA96

Tc (8C)

PLA100 PLA98 PLA96

DHc (J/g) na t1/2 (min)a DHc (J/g) na t1/2 (min) DHc (J/g) na t1/2 (min)

95 19.9 2.9 4.6 20.6 3.0 12.4 22.2 2.9 51.4100 21 2.9 3.0 21.0 2.7 9.3 21.6 2.7 36.1105 24.9 2.9 2.4 23.4 2.6 7.2 23.4 2.9 36.4110 23.2 2.8 3.4 25.8 3.1 9.7 24.7 2.9 56.8115 26.4 2.9 5.3 27.3 3.0 13.8120 30.7 3.0 7.6

a The crystallization half-time t1/2 was evaluated by (ln2/ka)1/n.



fined amorphous region was removed frombetween them. Enzymatic degradation seems notto affect the orientation of lamellae. After 72 hdegradation, the spherulites appeared moreeroded although the global morphology remainedmostly unchanged [Fig. 2(e)].

PLA98 Films. Incorporation of 2% D-lactyl unitsin polymer chains greatly affects the crystalliza-tion of PLA. The size of spherulites is ca. 200lm, i.e., much smaller than PLA100 ones. After24 h degradation, well-defined boundaries areobserved between spherulites and surroundingamorphous parts [Fig. 3(a)]. The spherulites

also exhibit numerous cracks along the radialdirection because of the removal of amorphousdomains entrapped between lamellar stacks.After 72 h, larger cracks are observed [Fig. 3(c)],and the crystalline fraction of spherulite wasfully visualized. Main lamella stacks in thespherulite appear closely packed, with largecracks between them, possibly because of thelower crystallization rate enabling the uncrys-tallizable chain segments to be sufficientlyexcluded outward.

A different morphology was observed after48 h degradation, as shown in Figure 3(b). Nonucleation center is distinguishable in the

Figure 2. ESEM photographs of PLA100 films crystallized at 135 8C for 2 h anddegraded by proteinase K (0.2 g/L) at 37 8C for various periods: (a) 0 h, (b) and (c)24 h, (d) 48 h, (e) 72 h.

964 HE ET AL.


spherulitic structure, which seems to be differentfrom two common models, i.e., branched symmet-ric spherulites with a central nucleus and spher-ulites through lamella growth of sheaf stage.This unique morphology could result from thefact that the nuclei are not located at the surface.

On the other hand, this finding suggests thatnormal 2-dimensional spherulitic morphology inan ultra thin film is confined by the surface andis not able to reflect actual 3-dimensional spheru-litic organization in bulk material.

PLA96, PLA94, and PLA92 Films. Spherulitesare hardly detectable in PLA96, PLA94, andPLA92 films since the undercooling degree is toolow for them to crystallize at 135 8C. Few smallirregular crystals were found for PLA96, whichappeared much less compact than those ofPLA100 and PLA 98, as shown in [Figure 4(a)],with some lamella branching outward. Nocrystal was found in PLA94 film Figure 4(b). Anetlike morphology was observed after 72 hdegradation, resulting from homogeneous sur-face erosion of amorphous material. Similarly,PLA92 appeared totally amorphous and showeda homogeneous surface degradation.

Figure 3. ESEM photographs of PLA98 films crys-tallized at 135 8C for 2 h and degraded by proteinaseK (0.2 g/L) at 37 8C for various periods: (a) 24 h,(b) 48 h, and (c) for 72 h.

Figure 4. ESEM photographs of PLA96 (a) andPLA94 (b) films crystallized at 135 8C for 2 h anddegraded by proteinase K (0.2 g/L) at 37 8C for 72 h.



Crystallization at 95 8C

There are no major differences between the sur-face morphologies of PLA100, PLA98, PLA96, andPLA94 films after 72 h enzymatic degradation,as shown in Figure 5. All of them present highnucleation density and small spherulite size. Noapparent amorphous domains are observedbetween spherulites. Some fibrils could bedetected as finite branching at the surface ofPLA100 film (indicted by arrows in [Fig. 5(a)].With D-lactyl units incorporated in polymerchains, the surface appeared more eroded due tohigher amorphous fraction [Fig. 5(a–d)]. Thelamella stacks are arranged irregularly withcurved shapes likely in sheaf morphology.

In the case of PLA92, 3-dimensional spher-ulites are observed after 6 h degradation, asshown in Figure 5 (e,f). Both free amorphousdomains between spherulites and confined onesbetween lamella stacks were eroded by protein-ase K. The lamella stacks appear rather coarsewhen compared with PLA100 ones probablybecause part of segments containing D-lactylunits is excluded outward the crystals and dis-posed as folds, tie chains, and chain ends on thelamella folding surface. Fisher et al. proposedthat the amount of D-lactyl units included intoL-lactyl crystal lattice depends strongly on theundercooling and D-lactyl units are preferen-tially rejected by crystallization kinetics.13 It is

Figure 5. ESEM photographs of PLA films crystallized at 95 8C for 2 h anddegraded by proteinase K (0.2 g/L) at 37 8C for 72 h (PLA92 degraded for 6 h): (a)PLA100, (b) PLA98, (c) PLA96, (d) PLA94, (e) and (f) PLA92.

966 HE ET AL.


Figure 6. ESEM photographs of PLA films crystallized under the same undercool-ing (Tm �30 8C) for 2 h and degraded by proteinase K (0.2 g/L) at 37 8C for 48 h:(a, b) PLA100, (c, d) PLA98, (e, f) PLA96, (g, h) PLA94, (i, j) PLA92.



of interest to note that PLA92 film was totallydegraded in 12 h.

Crystallization Under the Same Undercooling

As the values of Tm of PLA were indicated inTable 1, an isothermal condition of (Tm �30 8C)was introduced for PLA melt crystallization.The apparent melting temperature was used forthe undercooling approximation instead of theequilibrium melting temperature which was notdetermined. In Figure 6, the two typical kindsof crystalline morphology of PLA were shown foreach sample after 48 h proteinase K degrada-tion, i.e., one caused by nucleation on the sur-face (left column in Fig. 6) and the other byinner nucleation (right column in Fig. 6).

When nucleated on the surface, the lamellaestacks seem to be in a compact texture in edge-on mode, as shown in Figure 6(c,e). In contrast,lamellae stacks branch in a disordered morphol-ogy with inner nucleation and subsequentlyform a sphere [Fig. 6(h,j)]. The texture of spher-ulite is approximately composed of wider fibrilsas higher fraction of D-lactyl units is introducedinto PLA chains, with the crystalline fractionbranching like nerve fibers. The lateral dimen-

sions of lamellae are determined by the parame-ter d (d ¼ D/G, D is the diffusion coefficient andG is the growth rate) as proposed by Keith andPadden.33 As the value of G significantlydecreased with increasing D-lactyl content, dwould be higher which means that the lateraldimension of fibrils in spherulites should belarger. Therefore, a coarser organization oflamellae could be attained in PLA of higher frac-tion of D-lactyl units. This conclusion can simplybe applied to the results of PLA crystallizationat 135 8C (Fig. 2–4), with a constant D for vari-ous stereoregularities at the same crystallizationtemperature.

The various degraded PLA films were ana-lyzed by DSC in comparison with initial sam-ples. In Figure 7, PLA crystallinity indicated bythe melting enthalpy decreases with increasingD-lactyl content for both initial and degradedsamples. Consequently, the overall crystalgrowth rate is significantly affected by D-lactylincorporation at approximately the same under-cooling degree.

The same melting temperatures weredetected before and after degradation for eachsample. It is noted that SEC presents little dif-ference of molecular weights after degradation.

Figure 7. DSC thermograms of PLA bulk samples crystallized under the sameundercooling (Tm �30 8C) for 2 h before and after proteinase K degradation for 48 h.

968 HE ET AL.


This is in agreement with Iwata et al.’s results,which indicates that partial degradation at thechain-folding surfaces does not take place.18 Thestable chain-folding surfaces guarantee the foldsurface energy (re) remaining the same duringdegradation. Tm is determined by both l and reaccording to the expression of lamellar thickness(l) in LH theory,

l ¼ 2re � T0m

DH � T0m � Tm

� � ð2Þ

where DH is the ideal heat of fusion and T0m is

the equilibrium melting temperature. Therefore,the melting temperatures of the sampleswouldn’t change despite the removal of the con-fined amorphous fraction, since the lamellathickness and fold surface energy remain almostunchanged during degradation.

The crystallization peak appeared at lowertemperature with a higher exothermic value inthe heating process after degradation, as shownin case of PLA100 and PLA98. Besides, the en-thalpy recovery around the glass transition tem-perature was predominantly due to physicalaging process, especially in case of PLA92,PLA94, and PLA96 with higher amorphous frac-tion. Those results suggest that degradation pro-cess contributes to the improvement of chainorder in amorphous regions.

FTIR analyses provide further information onthe degradation properties. All five samples ex-hibit similar FTIR spectra. It has been reportedthat an absorption band at 921 cm�1 is charac-teristic of 103 helix conformation in a crystaland 955 cm�1 band is related to the amorphousstate of PLLA.35,36 As shown in Figure 8 forPLA98, a wide band at 921 cm�1 is detectedbefore enzymatic degradation. In contrast, twonew bands at 932 cm�1 and 905 cm�1 appearedafter 48 h degradation, together with the mainband at 921 cm�1. According to ESEM observa-tion, the confined amorphous part of spheruliteswas degraded by proteinase K and lamellaeretained their orientation. Therefore, enzymaticdegradation of the amorphous regions betweenlamellae stacks was supposed to influence theskeleton stretching and CH3 rocking in PLAlamellae and result in the splitting of the crys-talline band at 921 cm�1.

In addition, the enthalpy recovery due tophysical aging during degradation appears sig-nificantly around the glass transition tempera-

ture (Fig. 7). The physical aging effect could alsobe distinguished from FTIR spectra. In Figure8, a shoulder peak on the amorphous band at955 cm�1 presents after enzymatic degradation,which exhibits the partial order of local seg-ments affecting the skeletal vibration of amor-phous chains of PLA98. In contrast, no shoulderpeak was observed before degradation for nophysical aging happened.

The mass loss was calculated by comparingthe dry weight (mt) after degradation with theinitial weight (m0) according to the equation(%mass loss ¼ 100 3 (m0 � mt)/m0). PLA100 haslowest mass loss of 12%, followed by 34% forPLA98, 41% for PLA96, 51% for PLA94, and 60%for PLA92. It is well known that lower crystallin-ity and higher L-lactyl content lead to faster deg-radation by proteinase K. The fact that exhibitsPLA92 the fastest degradation rate indicates thecrystallinity is the predominant factor in thiscase.

CONCLUSIONS

DSC analysis shows that PLA homo- and stereo-copolymers with up to 8% of D-lactyl units pres-

Figure 8. Spectra in the 890–970 cm�1 region forcrystallized PLA80 (135 8C, 2 h) before and afterenzymatic degradation for 48 h.



ent the same crystallization mechanism, i.e.,bulk crystallization with heterogeneous nuclea-tion followed by 3-dimensional growth. The over-all rate of crystallization decreases with increas-ing D-lactyl units in PLA chains, but the maxi-mum crystallization rate was found at ca.105 8C for each PLA polymer. DHc shows littledependence on D-lactyl content at a given tem-perature for PLA100, PLA98 and PLA96, suggest-ing that the three PLA polymers have compara-ble crystallizable fractions.

ESEM study confirms that proteinase Kdegrades both the free and confined amorphousregions of melt crystallized PLA films. Lamellastacks in spherulites retain their orientationduring enzymatic degradation. Nucleus locationis proposed to be the main factor for the twowidely observed kinds of spherulite morpholo-gies in PLA films (ca. 80 lm). Specifically, 3-dimensional spherulites were directly observedafter degradation of PLA92 crystallized at 95 8C.At the same Tc, the incorporation of D-lactylunits affects the size and compactness of PLAspherulites. Under the same undercooling, thelateral dimension of crystalline fibrils increaseswith increasing the content of D-lactyl units.

The authors acknowledge the Pujiang Talents Project(No. 06PJ14010) of the Shanghai Science and Tech-nology Committee and Shanghai Leading AcademicDiscipline Project (No. B113) for financial support.

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Morphological investigation on melt crystallized polylactide homo- and stereocopolymers by enzymatic degradation with proteinase K