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Hydrolytic and enzymatic degradation of poly(trimethylene carbonate-co-d ,l -lactide) random copolymers with shape memory behavior
European Polymer Journal 46 (2010) 783–791
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European Polymer Journal journal homepage: www.elsevier.com/locate/europolj
Hydrolytic and enzymatic degradation of poly(trimethylene carbonate-co-D,L-lactide) random copolymers with shape memory behavior Jian Yang a,b, Feng Liu a, Liu Yang a,b, Suming Li a,b,* a b
Department of Materials Science, Fudan University, Shanghai 200433, China Max Mousseron Institute on Biomolecules, UMR CNRS 5247, University Montpellier I, 34060 Montpellier, France
a r t i c l e
i n f o
Article history: Received 27 August 2009 Received in revised form 9 December 2009 Accepted 13 December 2009 Available online 21 December 2009 Keywords: Enzymatic degradation Shape memory behavior Trimethylene carbonate Polylactide Hydrolytic degradation
a b s t r a c t A series of homo- and copolymers were synthesized by ring-opening polymerization of 1,3trimethylene carbonate and D,L-lactide, using low toxic Zn(Lac)2 as catalyst. The hydrolytic and enzymatic degradation of PTMC homopolymer and PTDLA copolymers was performed at 37 °C in pH 7.4 phosphate buffered saline or in pH 8.5 Tris buffer using proteinase K. Degradation was followed by using various analytical techniques such as NMR, GPC, DSC and ESEM. PTMC degrades extremely slowly by pure hydrolysis or in the presence of proteinase K. In contrast, PTDLA copolymers with different compositions degrade at various rates both in PBS and in enzyme solutions. The higher the LA content, the faster the degradation. LA units are preferentially degraded during hydrolytic degradation, indicating that ester bonds are more susceptible to hydrolytic cleavage than carbonate ones. Changes in surface morphology are observed during enzymatic degradation, in agreement with surface erosion process. The PTDLA11 copolymer with equivalent TMC/LA contents is highly elastic. Its residual strain is approximately 4% after the first cycle at a strain of 50%. The shape recovery ratio is up to 83%. Therefore, it is concluded that high molecular weight PTDLA copolymers are promising candidates for clinical applications in minimally invasive surgery. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Biodegradable polymers have been extensively investigated for temporary therapeutic applications such as surgical sutures, implanted medical devices, drug delivery systems, as well as tissue engineering scaffolds [1–3]. The rapid growth in this field has led to remarkable advances in materials science driven by the complex requirements of clinical applications. Multi-functionality such as biocompatibility and/or degradability combined with additional functions such as shape memory behavior for minimally invasive surgery has been considered [4–6]. The shape memory effect enables the implantation of a
* Corresponding author. Address: Max Mousseron Institute on Biomolecules, UMR CNRS 5247, University Montpellier I, 34060 Montpellier, France. E-mail address: [email protected] (S. Li). 0014-3057/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.eurpolymj.2009.12.017
bulky device in a compressed temporary shape through a small incision. Upon application of heat and thereby exceeding a certain switching temperature, the device changes to its original shape. Biodegradable polymers with shape memory property present great interest for biomedical applications such as self-expandable polymeric stents, bone fracture fixation devices, etc. A driving force such as physical or chemical cross-linking points is required in order to recover the initial shape after deformation and fixing. Shape memory polymers (SMP) can utilize glass transition [7], chain entanglements [6] and/or melting points [4,8] as the deformation/fixing temperature. SMPs display at least two phases, characterized by two distinct thermal transitions. The phase showing the higher transition temperature (associated with either a glass transition or a melting) acts as a physical cross-linker of the polymer chains and is responsible for the permanent shape. The second phase, with lower
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transition temperature, plays the role of a molecular switch. Above and below this switching temperature, the temporary shape is respectively formed and fixed. Polylactide (PLA) is perhaps the most important polyester in biomedical applications due to its many favorable characteristics, such as high strength, degradability, and biocompatibility [9,10]. Poly(L-lactide) (PLLA) is a semicrystalline polymer with a glass transition temperature (Tg) at 60 °C, while poly(D,L-lactide) (PDLLA) is amorphous with Tg 50 °C. PDLLA hydrolytically degrades much faster than PLLA due to different chain stereoregularities [11,12]. On the other hand, a protease from Tritirachium album, proteinase K, was found to be able to degrade PLA [13]. This enzyme preferentially degrades L units as opposed to D ones, PDLA being non-degradable [14]. The shape memory behavior of PLA has also been considered. The recovery ratio is relatively low and the recovery temperature is too high for uses in human body [15,16]. 1,3-Trimethylene carbonate (TMC) attracted much attention since it was used as a softening component together with glycolide to prepare copolymer sutures known as MaxonÒ [17]. Poly(trimethylene carbonate) (PTMC) is an amorphous elastomer with a glass transition at about 15 °C. PTMC exhibits good mechanical performance, including high flexibility and high tensile strength. It degrades in vivo by surface erosion without release of acidic species [18]. Degradation is also observed when PTMC is incubated in lipase solutions (from Thermomyces lanuginosus) [19]. The in vitro hydrolytic degradation of PTMC has been investigated at 37 or 60 °C. Degradation is very slow and is independent of the initial molecular weight and ionic strength of the conditioning medium [20]. Several studies have shown its potential as a biomaterial, in particular in soft tissue engineering [21– 23]. Copolymerization of polyesters with polycarbonates is a means to adjust the degradation rate and mechanical properties. Various copolymers of lactide and 1,3-trimetylene carbonate (TMC) have been reported [18,24–27]. These copoly(ester-carbonates) with outstanding tensile strength and flexibility have been investigated for applications as heart constructs and nerve regeneration guides [18,24], cartilage implants and wound dressing [25], sustained drug release carrier [26], and stent cover [27]. In our previous work, the hydrolytic degradation of a series of PLLA– PTMC copolymers has been investigated [28]. Various degradation behaviors and degradation rates were observed by varying the chemical composition, chain microstructure and morphology of PLLA–PTMC copolymers. However, no detailed study on the enzymatic degradation and shape memory properties of PDLLA–PTMC copolymers has been reported, so far. In this work, high molecular weight PTMC and PDLLA– PTMC were prepared by ring-opening copolymerization of D,L-lactide and 1,3-trimethylene carbonate. The hydrolytic degradation, enzymatic degradation, mechanical properties and shape memory behavior were investigated by mean of various analytical techniques such as proton nuclear magnetic resonance (1H NMR), gel permeation chromatography (GPC), differential scanning calorimetry (DSC), environmental scanning electronical micrography (ESEM), dynamic mechanical analysis (DMA) and Instron
tensile measurement. Water uptake, mass loss, composition and surface morphology of the different copolymers were evaluated as a function of degradation time. Particularly, a PDLLA–PTMC copolymer was selected to evaluate the shape recovery behavior at human body temperature for potential biomedical applications. 2. Experimental section 2.1. Materials D,L-lactic acid, sodium metal, 1,3-propanediol, diethyl carbonate, dimethylbenzene, tin powder and dibutyltin dilaurate were obtained from SCRC (China) and used as received. Dichloromethane, ethyl acetate, methanol and ethyl ester were of analytic grade and used without further purification. Hemizinc lactate (Zn(Lac)2) was supplied by Sigma. Proteinase K from T. album (P30 U/mg) in the form of lyophilized powder was purchased from Merck and used as received.
2.2. Synthesis and polymerization D,L-lactide was prepared by polycondensation of D,L-lactic acid, followed by thermal decomposition and cyclization. The crude product was purified 4 times by recrystallization in ethyl acetate prior to polymerization. TMC was prepared by using the procedure reported in literature [29]. In brief, 1,3-propanediol, diethyl carbonate, dibutyltin dilaurate, dimethylbenzene, and sodium metal were refluxed at 140 °C for 6 h. Ethanol, dimethylbenzene and residual diethyl carbonate were eliminated by distillation. Tin powder was then added, and the crude product was obtained by thermal decomposition at 180 °C. Purification was also performed 4 times prior to polymerization. PTMC and PDLLA–PTMC homo- and copolymers were synthesized by ring-opening polymerization of appropriate monomer feeds, using low toxic Zn(Lac)2 as catalyst (0.05 wt.%). The monomers and catalyst were charged in a silanized polymerization tube. After degassing, the polymerization tube was sealed under vacuum, and polymerization was allowed to proceed for 72 h at 140 °C. The resulting polymers were recovered by dissolution in dichloromethane and precipitation in methanol, followed by vacuum drying at room temperature up to constant weight.
2.3. Hydrolytic degradation The polymers were dissolved in dichloromethane and the resulting solutions (10 w/v%) were poured onto a glass plate. The solvent was allowed to evaporate overnight, followed by vacuum drying for 48 h. Square specimens with dimensions of approximately 10 10 0.3 mm were then cut from the films for degradation studies. The polymers were subjected to hydrolytic degradation by immersing the samples into small vials containing a stir bar and filled with 5 ml of pH 7.4 phosphate buffered saline (PBS). 0.02 wt.% NaN3 was added to the solution to prevent microbial growth. The vials were placed in incubator
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at 37 °C. The medium was renewed once a week. The samples were removed from the buffer solution at prescribed intervals and washed with deionized water. After wiping, the specimens were weighed and vacuum dried at room temperature for 1 week before analyses. The water uptake and mass loss were calculated according to the following equations:
water uptake ð%Þ ¼
mass loss ð%Þ ¼
ww wd 100 wd
wi wd 100 wi
ð1Þ
ð2Þ
where wi , ww , wd represent the initial weight, wet weight and dry weight of the samples, respectively. Three duplicate samples were used for each data point. 2.4. Enzymatic degradation Enzymatic degradation was performed at 37 °C in Tris buffer (0.05 M, pH 8.5) containing 0.2 mg/ml proteinase K. Sodium azide (0.02 wt.%) was added to inhibit the growth of microorganisms. The specimens (10 10 0.3 mm) were placed in vials field with 2 ml buffer solution which was renewed every 72 h to preserve the enzymatic activity. At preset time intervals, three duplicate specimens were withdrawn, washed thoroughly with distilled water and vacuum dried at room temperature for 1 week prior to various analyses.
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Dynamic mechanical measurements were carried out on hot pressed film samples using a Perkin-Elmer DMA Pyris-6 in tensile mode. The analyses were conducted at a frequency of 1 Hz and a heating rate of 2 °C/min, using a static force of 0.2 N and a dynamic force of 0.1 N, in the temperature range from 20 °C up to 20 °C above the Tg of the polymers. The shape memory behavior of the copolymer was quantitatively assessed by means of a combination of Instron 1121 dynamometer and TMA instrument in a twostep procedure, i.e., deformation and recovery, as described below. First, a hot pressed polymer strip with a width of 3.3 mm and a thickness of 0.66 mm was stretched at 37 °C up to 150% deformation by using Instron dynamometer, the strain rate being 20 mm/min and the gauge length 20 mm. Without releasing the sample from the grips, the deformed strip (i.e., the temporary shape) was fixed by quenching the sample to 0 °C with the aid of ice spray. Then the sample was removed from the dynamometer clamps, and immediately transferred to the second (recovery) step. Recovery was quantitatively assessed using a TMA analyzer in tensile mode and applying the following non-conventional procedure: the applied force was set to zero and the change of sample length was recorded during a heating scan at 2 °C/min from 0 to 70 °C. During this process, changes of stress (d), strain (e), and temperature (T) of the copolymer were recorded.
3. Results and discussion 2.5. Measurements 3.1. Synthesis and characterization The molecular weights of the polymers were determined on a Waters apparatus equipped with an RI detector. THF was used as the mobile phase at a flow rate of 1.0 ml/min. 20 ll of 1.0 w/v% solution was injected for each analysis. Calibration was accomplished with polystyrene standards (Polysciences, Warrington, PA). The copolymer composition was determined by 1H NMR spectroscopy. The 1H NMR spectra of the copolymers were recorded at a Bruker AV500 NMR spectrometer at ambient temperature, using DMSO as solvent, and TMS as the internal reference. The spectra were obtained with a FID resolution of 0.245 Hz/point, corresponding to a sweep width of 8 kHz, acquisition time was 2.04 s. Differential scanning calorimetry (DSC) was registered with a Perkin-Elmer DSC Pyris-6 instrument, at a heating rate of 10 °C/min. About 10 mg of product was used for each analysis. The glass transition temperature (Tg) was taken at the midpoint of the transition zone on the second heating scan. The surface of degraded samples of all the polymers was examined by using a Philips XL30 ESEM Tungsten Scanning Electron Microscope under reduced pressure (5 Torr). Stress–strain measurements were carried out on strips die-cut from solvent-cast sheets by means of an Instron 1121 tensile testing machine at room temperature. Strips with 0.5 mm thickness and 3 mm width were used. The cross-head speed was 40 mm/min, and the gauge length was 20 mm. Six measurements were performed for each polymer.
PTMC homopolymer and PTMC–PDLLA copolymers with TMC/DL-LA feed ratios of 2/1, 1/1 and 1/2 denoted as PTDLA21, PTDLA11 and PTDLA12 were synthesized by bulk ring-opening polymerization of 1,3-trimethylene carbonate and D,L-lactide. Zn(Lac)2 was used as catalyst instead of stannous octoate or other organometallic catalysts which are more or less cytotoxic. The 1H NMR spectrum of PTDLA11 copolymer is shown in Fig. 1. Signals in the 4.9–5.2 ppm zone and at 1.5 ppm are assigned to –CH– and –CH3 of lactyl units, and those at 4.2 and 2.0 ppm to –CH2 of TMC units, respectively [30]. Interestingly, signals in the 4.9–5.2 ppm zone can be divided in two groups: the downfield group around 5.2 ppm belongs to main chain lactyl units, and the upfield group around 5.0 ppm is assigned to lactyl units linking to TMC units. The presence of many TMC-linking lactyl units indicates the random nature of the chain structure. The composition of the copolymers is calculated from the integrations of the signal at 4.0–4.2 ppm for TMC units and that at 5.0– 5.3 ppm for DL-LA units. As shown in Table 1, the [TMC]/ [LA] molar ratio is 67/33 and 48/52 for PTDLA21 and PTDLA11, respectively, which is very close to the feed ratio. In contrast, the TMC content in PTDLA12 is lower than in the feed, probably due to the lower reactivity of TMC monomer as compared to lactide, as reported in literature [31]. GPC was used to evaluate the molecular weights and polydispersity of the polymers. The number average molecular weights (Mn) of PTMC, PTDLA11 and PTDLA12
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Fig. 1. 1H NMR spectrum of PTDLA11 copolymer in DMSO.
are all above 100,000, whereas the Mn of PTDLA21 is slightly lower (Table 1). On the other hand, the polydispersit index (Ip = Mw/Mn) is in the range of 1.7–1.9. Therefore, Zn(Lac)2 is an efficient catalyst for the copolymerization of TMC and LA monomers. The thermal properties of the polymers were determined by DSC. All the polymers are intrinsically amorphous as a single glass transition is detected, in agreement with the random chain structure. The Tg of PTMC is detected at 10.6 °C, indicating that it is a rubbery polymer at room temperature [32]. The Tg of PTDLA copolymers increases with increasing D,L-LA content (Table 1), in agreement with the fact the Tg of PDLLA (50 °C) is much higher than that of PTMC.
slowly as no mass loss is detected, in agreement with the literature [33]. In contrast, PTDLA copolymers appear much more hydrolytically degradable. No mass loss is observed for PTDLA21 and PTDLA11 during the first 9 weeks, while the mass loss of PTDLA12 reaches 3.4% probably due to the release of low molecular weight species such as residual monomers [34]. Mass loss slightly increases for PTDLA21 and PTDLA11 to reach 3.5% after 21 weeks. Beyond, mass loss rapidly increases for the three copolymers. The final mass loss is 49% for PTDLA21, 49% for PTDLA11 and 50% for PTDLA12 at week 53. Apparently, PTDLA copolymers degrade much faster than PTMC homopolymer since ester bonds are more susceptible to hydrolysis than carbonate bonds as previously observed [20]. The hydrolysis rate of polyesters is influenced by the access to water for the labile ester bonds, Water uptake occurs when a polymer sample is immersed in an aqueous medium even though the polymer is hydrophobic [33]. Fig. 2b presents the water uptake profiles of the polymers during hydrolytic degradation. PTMC homopolymer appears very hydrophobic with only 3% of water uptake after
3.2. Hydrolytic degradation The hydrolytic degradation of the polymers was performed in pH 7.4 PBS at 37 °C. Fig. 2a shows the mass loss profiles of the polymers as a function of degradation time up to 53 weeks. PTMC homopolymer degrades extremely
Table 1 composition, molecular weight, thermal and mechanical properties of PTLA copolymers.
a b c d
Samples
Feed ratio (TMC/LA)
Molar ratioa (TMC/LA)
Tg (°C)
PTMC PTDLA21 PTDLA11 PTDLA12
100/0 67/33 50/50 33/67
100/0 67/33 48/52 18/82
10.6 11.4 22.4 44
Calculated from 1H NMR. Tensile strength. Stain at break. Young’s modulus.
GPC Mn
DPI
114,000 88,000 104,000 119,000
1.9 1.8 1.8 1.7
db (MPa)
ebreakc (%)
Ed (MPa)
1.4 0.41 0.57 0.73
720 650 620 540
4.7 2.0 2.1 2.4
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Fig. 4. Changes of LA content in the PTDLA copolymers during hydrolytic degradation.
Fig. 2. (a) Mass loss and (b) water uptake of the copolymers during hydrolytic degradation. Three duplicate samples were used for each data point.
21 weeks. PTDLA21 and PTDLA11 present slightly higher water uptake which reaches 8% and 9%, respectively. In
Fig. 3. Molecular weight profiles of the polymers during hydrolytic degradation as measured by GPC.
contrast, nearly 27% of water uptake is obtained for PTDLA12 in the same period. Higher water uptake is observed for PTDLA copolymers as compared to PTMC because degradation of PTDLA leads to formation of hydrophilic OH and COOH endgroups. GPC was used to monitor changes of molecular weights of the polymers during degradation. Fig. 3 presents the normalized Mn changes as a function of degradation time. The Mn of PTMC homopolymer appears almost unchanged during the degradation period up to 53 weeks, in agreement with the fact that this polymer is not degradable by pure hydrolysis. In contrast, the copolymers degrade much faster than PTMC. Mn decrease is 66% after 16 weeks for PTDLA12, 57% for PTDLA11 and 38% for PTDLA21. Therefore, the higher the TMC content, the slower the Mn decrease, in agreement with the non-hydrodegradability of PTMC. Beyond, Mn changes tend to converge for three PTDLA copolymers. Compositional changes of the copolymers during degradation were obtained from 1H NMR (Fig. 4). The composition remains almost unchanged during the first 21 weeks, in agreement with the fact that no significant mass loss is detected. Beyond, LA content decreases from 33% to 22% for PTDLA21, from 51% to 43% for PTDLA11, and from 82% to 78% for PTDLA12. These findings indicate that LA units are preferentially degraded in amorphous PTDLA copolymers, as previously reported [28]. However, the compositional changes are less than what could be expected from the mass loss (50%), indicating that TMC units are degradable in PTDLA copolymers although PTMC homopolymer is not degradable. PTDLA12 exhibits the smallest LA content decrease since it initially contains the largest LA content (82%). Comparison of the Mn decrease, mass loss data and compositional changes allows to better understand the hydrolytic degradation behaviors of the copolymers. Hydrolytic cleavage of copolymer chains immediately starts after immersion in the PBS, leading to rapid Mn decrease. Nevertheless, significant mass loss is observed much later, i.e., beyond 21 weeks. In fact, the Mn of the copolymers decreases from nearly 20,000 to 6000 from
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Fig. 5. (a) Mass loss and (b) water uptake of the polymers during enzymatic degradation. Three duplicate samples were used for each data point.
21 to 35 weeks. Low Mn species are thus formed and released into the medium, which results in rapid mass loss. LA units are preferentially degraded along the copolymer chains, leading to compositional changes.
the LA content, the faster the degradation in the presence of proteinase K which cannot degrade PTMC homopolymer. Fig. 5b illustrates the water uptake curves during enzymatic degradation of the polymers. PTDLA12 with LA content of 82% presents a gradual increase in water uptake, up to 15.7% after 264 h, whereas the water uptake of PTMC, PTDLA21 and PTDLA11 is approximately 3%. These findings confirm the higher bulk hydrophilicity of PTDLA12, in agreement with the data from hydrolytic degradation. The changes of molecular weights and compositions are followed by using GPC and NMR. The composition of the copolymers remains almost unchanged during the degradation period. The only exception is PTDLA12 whose LA content slightly decreases from 82% to 78% after 216 h, in contrast to the hydrolytic degradation data. This finding could be attributed to the very important mass loss of PTDLA12 (nearly 90%) after 216 h enzymatic degradation. On the other hand, the Mn of the PTDLA copolymers slightly decreases, while that of PTMC remains almost the same during enzymatic degradation. It is well known that enzymatic degradation is a surface erosion process which hardly affects the bulk properties, but hydrolytic degradation still takes place in the bulk. Similar findings have been reported in the case of PCL–PLLA block or random copolymers during degradation by pseudomonas lipase [36,37]. ESEM measurements were performed to examine the surface morphology of the polymers during enzymatic degradation. No changes are detected after degradation for PTMC due to the absence of degradation. Similarly, PTDLA21 presents little changes after degradation in agreement with the small mass loss (11%). In contrast, PTDLA11 and PTDLA12 appear largely eroded in the presence of proteinase K. The original samples exhibit a smooth or slightly rugged surface as illustrated in Fig. 6a and d for PTDLA11 and PTDLA12, respectively. However, a highly porous structure is detected for PTDLA11 (Fig. 6b and c) degraded after 120 and 216 h, and for PTDLA12 (Fig. 6e and f) after 120 and 216 h. Similar findings were previously reported for PLA [13,38]. Therefore, PTDLA copolymers are degradable in the presence of proteinase K, in particular for those with high LA contents. 3.4. Mechanical properties
3.3. Enzymatic degradation Proteinase K is largely used for enzymatic degradation of PLA-based homo- and copolymers [13,14,35]. The enzyme-catalyzed degradation of PTDLA copolymers was carried out using proteinase K in pH 8.5 Tris buffer at 37 °C. PTMC homopolymer was taken as control. Fig. 5a shows the mass loss curves of PTMC and PTDLA copolymers during degradation. PTMC appears non-degradable in the presence of proteinase K as no significant mass loss is detected after 264 h. In contrast, the copolymers exhibit various degradation rates. Mass loss of PTDLA21 increases slowly but constantly to reach 11% after 264 h. PTDLA11 presents faster mass loss which reaches nearly 37% in the same period. The highest mass loss (91%) is obtained for PTDLA12 containing 82% of LA units. The difference should be ascribed to the compositions of copolymers. The higher
The mechanical properties of high molecular weight PTMC homopolymer and PTDLA copolymers were evaluated by using an Instron 1121 testing machine with a tensile rate of 40 mm/min at room temperature. The tensile strength (d), strain at break (ebreak) and Young’s modulus (E) are listed in Table 1. PTMC exhibits an ebreak value of 719%, in agreement with its elastomeric behavior. Copolymerization of TMC with D,L-LA results in decrease of the ebreak. The higher the LA content, the lower the ebreak. Nevertheless, the ebreak of the copolymers are all above 500%. The tensile strength and Young’s modulus of PTMC are 1.4 and 4.7 MPa, respectively. Copolymerization of TMC with D,L-LA results in decrease of tensile strength and Young’s modulus due to disordered chain structure. On the other hand, an increase of both parameters is observed with increasing LA content among the three PTDLA
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Fig. 6. ESEM micrographs of PTDLA11 (a–c) and PTDLA12 (d–f) copolymers after 0, 120 and 216 h enzymatic degradation.
Fig. 7. Tensile dynamic mechanical analysis (DMA) measurements for PTMC homopolymer and PTDLA copolymers.
copolymers. This can be assigned to the fact that poly(D,Llactide) homopolymer is a thermoplastic with much higher tensile strength and Young’s modulus than PTMC homopolymer. DMA was used to determine the storage modulus (E0 ) and tan d of all the polymers as a function of temperature (Fig. 7). PTDLA12 exhibits the highest storage modulus, nearly 3.5 GPa at 10 °C. The storage modulus slightly decreases with increasing temperature, and a strong decrease is observed in the range from 30 to 45 °C. On the other hand, the storage modulus decreases with increasing TMC content, PTMC homopolymer exhibiting the lowest E0 value. The tan d of the copolymers exhibits a peak value corresponding to the glass transition, which corroborates with data obtained from DSC analysis. PTDLA11 presents the highest tan d value of nearly 2.5, indicating its high energy storage and recovery ability. Moreover, the Tg of PTDLA11 is detected around the room temperature. PTDLA11 is thus selected for further investigations.
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Fig. 9. Shape memory experiment on a strip of PTDLA11 copolymer: the permanent shape (bend, a); the temporary shape (spiral, b); the recovery process at 37 °C approximately (c) after 2 s and (d) after 10 s.
3.5. Shape memory behavior of PTDLA11
Fig. 8. Stress–strain properties of the PTDLA11 copolymer upon cycle loading (a) cycles 1–20, and (b) cycles 21–23 (after 2 h relaxation).
The shape memory properties of the PTDLA11 copolymer with a Tg of 22.4 °C were evaluated using a hot pressed film between two selected temperature (37 and 0 °C). Fig. 9 shows the permanent shape, the temporary shape, and the recovered shape after heat treatment. Firstly, PTDLA11 (a bend strip, Fig. 9a) were stretched and wrapped around a rod in an incubator at 37 °C to reach an elongation higher than 150% (step 1), and then it was rapidly cooled down to 0 °C (step 2), the temporary shape (spiral, Fig. 9b) is obtained. The shape recovery process (step 3) is performed at 37 °C as shown in Fig. 10c and 10d. After 2 s and 10 s, the strip progressively recovers its permanent shape. Fig. 10 summarizes the results of a typical shape memory experiment in a three-dimensional graph. Path 1 from position A to position B is a stress–strain curve at a
Fig. 8 shows the typical stress–strain curves of PTDLA11 recorded for different loading cycles. PTDLA11 appears highly elastic. The residual strain is approximately 4% after the first cycle at a strain of 50%. Nearly 80% recovery is obtained after 20 cycles. The cycle loading tests show that the mechanical properties of the copolymer are affected by repeated loading, especially after the first and second cycles. The hysteresis shape becomes narrower during the following cycles. Every cycle loading test results in a mechanical hysteresis due to the viscoelastic nature of the polymer, while the difference between the ascending curves of two successive cycles can be explained by the non-reversible reorientation of macromolecular chains. After 20 cycles, a recovery period of 2 h was applied and 3 more cycles were recorded. At the beginning of the 21st cycle, PTDLA11 exhibits a permanent set of approximately 6%, i.e., much lower than the value of 20% obtained after 20 cycles. This finding can be assigned to relaxation of chains during the 2-h recovery period. After the 21st cycle, similar stress–strain curves are observed. Meanwhile, the residual strain in the 21st cycle is up to approximately 11%, showing that repeated loading and relaxation have an important effect on the structure of the linear polymer.
Fig. 10. Thermomechanical experiment on a strip of PTDLA11 copolymer: (1) stress–strain curve at 37 °C (strain rate, 20 mm/min); (2) recovery process (heating rate, 2 °C/min).
J. Yang et al. / European Polymer Journal 46 (2010) 783–791
constant temperature (37 °C) preformed in the dynamometer, the 150% strained strip is taken as the temporary shape and corresponds to the maximum strain (em). After quenching down to 0 °C and the load withdrawn, the sample can recover from position C to position D at a heating rate of 2 °C/min as path 2 performed in TMA. The recorded residual strain (er) of PTDLA11 after one cycle is 25%. Because the deformation recovery ability of the sample can be quantified by the shape recovery ratio parameter (Rr), defined as follows:
Rr ¼ ðem er Þ=em
ð3Þ
where em is the strain imposed to the strip at 37 °C and er is the measured residual strain. The Rr of PTDLA11 is found to be 83%. The initial recovery temperature taken from the intersection of tangent and baseline prior to recovery is 23 °C, i.e., very close to its Tg. It is commonly accepted that the polymer with shape memory behavior should possess two components at the molecular level: a fixing phase constituted by physical or chemical cross-links to fix the shape and a reversible phase to provide the elastomeric deformation. Thermo-responsive SMPs are generally networks. However, Zini et al. [6] reported the linear PLLA–PGA–PTMC terpolymers exhibit shape memory behavior by chain entanglements. It is also the case of linear PTDLA11 copolymer with high molecular weight. Time and temperature strongly influence the shape memory behavior of amorphous copolymers. It appears that temperature and deformation time must be kept as low as possible to restrain disentanglement of polymeric chains leading to irreversible flow. 4. Conclusions The hydrolytic and enzymatic degradation of PTMC homopolymer and PTDLA copolymers were investigated under in vitro conditions. PTMC homopolymer degrades extremely slowly by pure hydrolysis or in the presence of proteinase K. In contrast, PTDLA copolymers with different compositions degrade at various rates both in PBS and in enzyme solutions. The higher the LA content, the faster the degradation. LA units are preferentially degraded during hydrolytic degradation, indicating that ester bonds are more susceptible to hydrolytic cleavage than carbonate ones. Changes in surface morphology are observed during enzymatic degradation, in agreement with surface erosion process. The PTDLA11 copolymer with equivalent TMC/LA contents is highly elastic with outstanding shape memory property. Therefore, high molecular weight PTDLA copolymers are promising candidates for clinical applications in minimally invasive surgery. Acknowledgements The authors acknowledge the National Basic Research Program of China (973 Program No. 2007CB935801), the National Natural Science Foundation of China (No.
791
50873030), and the Shanghai Leading Academic Discipline Project (No. B113) for financial support. References [1] Dunn RL. Clinical applications and update on the poly (a-hydroxyacids). In: Hollinger JO, editor. Biomedical applications of synthetic biodegradable polymers. Boca Raton: CRC Press; 1995. p. 17–31. [2] Li SM. J Biomed Mater Res Appl Biomater 1999;48:142–53. [3] Li SM, Vert M. Biodegradable polymers: polyesters. In: Mathiowitz E, editor. Encyclopedia of controlled drug delivery. New York: John Wiley & Sons; 1999. p. 71–93. [4] Lendlein A, Langer R. Science 2002;296:1673–6. [5] Chen MC, Tsai HW, Chang Y, Lai WY, Mi FL, Liu CT, et al. Biomacromolecules 2007;8:2774–80. [6] Zini E, Scandola M, Dobrzynski P, Kasperczyk J, Bero M. Biomacromolecules 2007;8:3661–7. [7] Choi N-Y, Lendlein A. Soft Matter 2007;3:901–9. [8] Lee KM, Knight PT, Chung T, Mather PT. Macromolecules 2008;41:4730–8. [9] Tsuji H. Macromol Biosci 2005;5:569–97. [10] (a) Jain RA. Biomaterials 2000;2:2475–90; (b) Middleton JC, Tipton AJ. Biomaterials 2000;21:2335–46; (c) Albertsson A-C, Varma IK. Biomacromolecules 2003;4:1466–86. [11] Li SM, Garreau H, Vert M. J Mater Sci Mater Med 1990;1:123–30. [12] Grizzi I, Garreau H, Li SM, Vert M. Biomaterials 1995;16:305–11. [13] Li SM, Girard A, Garreau H, Vert M. Polym Degrad Stab 2001;71:61–7. [14] Reeve MS, McCarthy SP, Downey MJ, Gross RA. Macromolecules 1994;27:825–31. [15] Lu XL, Cai W, Gao ZY, Zhao LC. Mater Sci Eng A 2006;438– 440:857–61. [16] Zheng X, Zhou S, Li X, Weng J. Biomaterials 2006;27:4288–95. [17] Katz AR, Mukherjee DP, Kaganov AL, Gordon S. Surg Gynecol Obstet 1985;161:213–22. [18] Pêgo AP, Van luyn MJA, Brouwer LA, Van Wachem PB, Poot AA, Grijpma DW, et al. J Biomed Mater Res 2003;67A:1044–54. [19] Zhang Z, Zou SG, Vancso GJ, Grijpma DW, Feijen J. Biomacromolecules 2005;6:3404–9. [20] Albertsson A-C, Eklund M. J Appl Polym Sci 1995;57:87–103. [21] Matsuda T, Kwon I-K, Kidoaki S. Biomacromolecules 2004;5: 295–305. [22] Vandermeulen G, Rouxhet L, Arien A, Brewster ME, Préat V. Int J Pharm 2006;309:234–40. [23] Dinarvand R, Alimorad MM, Amanlou M, Akbari H. J Biomed Mater Res 2005;75A:185–91. [24] Pêgo AP, Poot AA, Grijpma DW, Feijen J. J Controlled Release 2003;87:69–79. [25] Andronova N, Albetsson AC. Biomacromolecules 2006;7:1489–95. [26] Zhu KJ, Zhang JX, Wang C, Yasuda H, Ichimaru A, Yamamoto K. J Microencapsulation 2003;20:731–43. [27] Asplund B, Sperens J, Mathisen T, Hilborn J. J Biomat Sci Polym Ed 2006;17:615–30. [28] Hua JJ, Gebarowska K, Dobrzynski P, Kasperczyk J, Wei J, Li SM. J Polym Sci A Polym Chem 2009;47:3869–79. [29] Hu B, Zhuo RX, Fan CL. Chem Reagents 1998;20(6):355–6. [30] Pospiech D, Komber H, Jehnichen D, Häussler L, Eckstein K, Scheibner H, et al. Biomacromolecules 2005;6:439–46. [31] Ruckenstein E, Yuan Y. J Appl Polym Sci 1998;69:1429–34. [32] Zhu KJ, Hendren RW, Jensen K, Jensen CG. Macromolecules 1991;24:1736–40. [33] (a) Pêgo AP, Poot AA, Grijpma DW, Feijen J. J Biomater Sci Polym Ed 2001;12:35–53; (b) Pêgo AP, Poot AA, Grijpma DW, Feijen J. Macromol Biosci 2002;2:411–9. [34] Stridsberg K, Albertsson A-C. Polymer 2000;41:7321–30. [35] Tsutsumi C, Nakagawa K, Shirahama H, Yasuda H. Macromol Biosci 2002;2:223–32. [36] Li SM, Liu LJ, Garreau H, Vert M. Biomacromolecules 2003;4:372–7. [37] Zhao ZX, Yang L, Hu YF, He Y, Wei J, Li SM. Polym Degrad Stab 2007;92:1769–77. [38] Li SM, Tenon M, Garreau H, Braud C, Vert M. Polym Degrad Stab 2000;67:85–90.
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European Polymer Journal journal homepage: www.elsevier.com/locate/europolj
Hydrolytic and enzymatic degradation of poly(trimethylene carbonate-co-D,L-lactide) random copolymers with shape memory behavior Jian Yang a,b, Feng Liu a, Liu Yang a,b, Suming Li a,b,* a b
Department of Materials Science, Fudan University, Shanghai 200433, China Max Mousseron Institute on Biomolecules, UMR CNRS 5247, University Montpellier I, 34060 Montpellier, France
a r t i c l e
i n f o
Article history: Received 27 August 2009 Received in revised form 9 December 2009 Accepted 13 December 2009 Available online 21 December 2009 Keywords: Enzymatic degradation Shape memory behavior Trimethylene carbonate Polylactide Hydrolytic degradation
a b s t r a c t A series of homo- and copolymers were synthesized by ring-opening polymerization of 1,3trimethylene carbonate and D,L-lactide, using low toxic Zn(Lac)2 as catalyst. The hydrolytic and enzymatic degradation of PTMC homopolymer and PTDLA copolymers was performed at 37 °C in pH 7.4 phosphate buffered saline or in pH 8.5 Tris buffer using proteinase K. Degradation was followed by using various analytical techniques such as NMR, GPC, DSC and ESEM. PTMC degrades extremely slowly by pure hydrolysis or in the presence of proteinase K. In contrast, PTDLA copolymers with different compositions degrade at various rates both in PBS and in enzyme solutions. The higher the LA content, the faster the degradation. LA units are preferentially degraded during hydrolytic degradation, indicating that ester bonds are more susceptible to hydrolytic cleavage than carbonate ones. Changes in surface morphology are observed during enzymatic degradation, in agreement with surface erosion process. The PTDLA11 copolymer with equivalent TMC/LA contents is highly elastic. Its residual strain is approximately 4% after the first cycle at a strain of 50%. The shape recovery ratio is up to 83%. Therefore, it is concluded that high molecular weight PTDLA copolymers are promising candidates for clinical applications in minimally invasive surgery. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Biodegradable polymers have been extensively investigated for temporary therapeutic applications such as surgical sutures, implanted medical devices, drug delivery systems, as well as tissue engineering scaffolds [1–3]. The rapid growth in this field has led to remarkable advances in materials science driven by the complex requirements of clinical applications. Multi-functionality such as biocompatibility and/or degradability combined with additional functions such as shape memory behavior for minimally invasive surgery has been considered [4–6]. The shape memory effect enables the implantation of a
* Corresponding author. Address: Max Mousseron Institute on Biomolecules, UMR CNRS 5247, University Montpellier I, 34060 Montpellier, France. E-mail address: [email protected] (S. Li). 0014-3057/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.eurpolymj.2009.12.017
bulky device in a compressed temporary shape through a small incision. Upon application of heat and thereby exceeding a certain switching temperature, the device changes to its original shape. Biodegradable polymers with shape memory property present great interest for biomedical applications such as self-expandable polymeric stents, bone fracture fixation devices, etc. A driving force such as physical or chemical cross-linking points is required in order to recover the initial shape after deformation and fixing. Shape memory polymers (SMP) can utilize glass transition [7], chain entanglements [6] and/or melting points [4,8] as the deformation/fixing temperature. SMPs display at least two phases, characterized by two distinct thermal transitions. The phase showing the higher transition temperature (associated with either a glass transition or a melting) acts as a physical cross-linker of the polymer chains and is responsible for the permanent shape. The second phase, with lower
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transition temperature, plays the role of a molecular switch. Above and below this switching temperature, the temporary shape is respectively formed and fixed. Polylactide (PLA) is perhaps the most important polyester in biomedical applications due to its many favorable characteristics, such as high strength, degradability, and biocompatibility [9,10]. Poly(L-lactide) (PLLA) is a semicrystalline polymer with a glass transition temperature (Tg) at 60 °C, while poly(D,L-lactide) (PDLLA) is amorphous with Tg 50 °C. PDLLA hydrolytically degrades much faster than PLLA due to different chain stereoregularities [11,12]. On the other hand, a protease from Tritirachium album, proteinase K, was found to be able to degrade PLA [13]. This enzyme preferentially degrades L units as opposed to D ones, PDLA being non-degradable [14]. The shape memory behavior of PLA has also been considered. The recovery ratio is relatively low and the recovery temperature is too high for uses in human body [15,16]. 1,3-Trimethylene carbonate (TMC) attracted much attention since it was used as a softening component together with glycolide to prepare copolymer sutures known as MaxonÒ [17]. Poly(trimethylene carbonate) (PTMC) is an amorphous elastomer with a glass transition at about 15 °C. PTMC exhibits good mechanical performance, including high flexibility and high tensile strength. It degrades in vivo by surface erosion without release of acidic species [18]. Degradation is also observed when PTMC is incubated in lipase solutions (from Thermomyces lanuginosus) [19]. The in vitro hydrolytic degradation of PTMC has been investigated at 37 or 60 °C. Degradation is very slow and is independent of the initial molecular weight and ionic strength of the conditioning medium [20]. Several studies have shown its potential as a biomaterial, in particular in soft tissue engineering [21– 23]. Copolymerization of polyesters with polycarbonates is a means to adjust the degradation rate and mechanical properties. Various copolymers of lactide and 1,3-trimetylene carbonate (TMC) have been reported [18,24–27]. These copoly(ester-carbonates) with outstanding tensile strength and flexibility have been investigated for applications as heart constructs and nerve regeneration guides [18,24], cartilage implants and wound dressing [25], sustained drug release carrier [26], and stent cover [27]. In our previous work, the hydrolytic degradation of a series of PLLA– PTMC copolymers has been investigated [28]. Various degradation behaviors and degradation rates were observed by varying the chemical composition, chain microstructure and morphology of PLLA–PTMC copolymers. However, no detailed study on the enzymatic degradation and shape memory properties of PDLLA–PTMC copolymers has been reported, so far. In this work, high molecular weight PTMC and PDLLA– PTMC were prepared by ring-opening copolymerization of D,L-lactide and 1,3-trimethylene carbonate. The hydrolytic degradation, enzymatic degradation, mechanical properties and shape memory behavior were investigated by mean of various analytical techniques such as proton nuclear magnetic resonance (1H NMR), gel permeation chromatography (GPC), differential scanning calorimetry (DSC), environmental scanning electronical micrography (ESEM), dynamic mechanical analysis (DMA) and Instron
tensile measurement. Water uptake, mass loss, composition and surface morphology of the different copolymers were evaluated as a function of degradation time. Particularly, a PDLLA–PTMC copolymer was selected to evaluate the shape recovery behavior at human body temperature for potential biomedical applications. 2. Experimental section 2.1. Materials D,L-lactic acid, sodium metal, 1,3-propanediol, diethyl carbonate, dimethylbenzene, tin powder and dibutyltin dilaurate were obtained from SCRC (China) and used as received. Dichloromethane, ethyl acetate, methanol and ethyl ester were of analytic grade and used without further purification. Hemizinc lactate (Zn(Lac)2) was supplied by Sigma. Proteinase K from T. album (P30 U/mg) in the form of lyophilized powder was purchased from Merck and used as received.
2.2. Synthesis and polymerization D,L-lactide was prepared by polycondensation of D,L-lactic acid, followed by thermal decomposition and cyclization. The crude product was purified 4 times by recrystallization in ethyl acetate prior to polymerization. TMC was prepared by using the procedure reported in literature [29]. In brief, 1,3-propanediol, diethyl carbonate, dibutyltin dilaurate, dimethylbenzene, and sodium metal were refluxed at 140 °C for 6 h. Ethanol, dimethylbenzene and residual diethyl carbonate were eliminated by distillation. Tin powder was then added, and the crude product was obtained by thermal decomposition at 180 °C. Purification was also performed 4 times prior to polymerization. PTMC and PDLLA–PTMC homo- and copolymers were synthesized by ring-opening polymerization of appropriate monomer feeds, using low toxic Zn(Lac)2 as catalyst (0.05 wt.%). The monomers and catalyst were charged in a silanized polymerization tube. After degassing, the polymerization tube was sealed under vacuum, and polymerization was allowed to proceed for 72 h at 140 °C. The resulting polymers were recovered by dissolution in dichloromethane and precipitation in methanol, followed by vacuum drying at room temperature up to constant weight.
2.3. Hydrolytic degradation The polymers were dissolved in dichloromethane and the resulting solutions (10 w/v%) were poured onto a glass plate. The solvent was allowed to evaporate overnight, followed by vacuum drying for 48 h. Square specimens with dimensions of approximately 10 10 0.3 mm were then cut from the films for degradation studies. The polymers were subjected to hydrolytic degradation by immersing the samples into small vials containing a stir bar and filled with 5 ml of pH 7.4 phosphate buffered saline (PBS). 0.02 wt.% NaN3 was added to the solution to prevent microbial growth. The vials were placed in incubator
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at 37 °C. The medium was renewed once a week. The samples were removed from the buffer solution at prescribed intervals and washed with deionized water. After wiping, the specimens were weighed and vacuum dried at room temperature for 1 week before analyses. The water uptake and mass loss were calculated according to the following equations:
water uptake ð%Þ ¼
mass loss ð%Þ ¼
ww wd 100 wd
wi wd 100 wi
ð1Þ
ð2Þ
where wi , ww , wd represent the initial weight, wet weight and dry weight of the samples, respectively. Three duplicate samples were used for each data point. 2.4. Enzymatic degradation Enzymatic degradation was performed at 37 °C in Tris buffer (0.05 M, pH 8.5) containing 0.2 mg/ml proteinase K. Sodium azide (0.02 wt.%) was added to inhibit the growth of microorganisms. The specimens (10 10 0.3 mm) were placed in vials field with 2 ml buffer solution which was renewed every 72 h to preserve the enzymatic activity. At preset time intervals, three duplicate specimens were withdrawn, washed thoroughly with distilled water and vacuum dried at room temperature for 1 week prior to various analyses.
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Dynamic mechanical measurements were carried out on hot pressed film samples using a Perkin-Elmer DMA Pyris-6 in tensile mode. The analyses were conducted at a frequency of 1 Hz and a heating rate of 2 °C/min, using a static force of 0.2 N and a dynamic force of 0.1 N, in the temperature range from 20 °C up to 20 °C above the Tg of the polymers. The shape memory behavior of the copolymer was quantitatively assessed by means of a combination of Instron 1121 dynamometer and TMA instrument in a twostep procedure, i.e., deformation and recovery, as described below. First, a hot pressed polymer strip with a width of 3.3 mm and a thickness of 0.66 mm was stretched at 37 °C up to 150% deformation by using Instron dynamometer, the strain rate being 20 mm/min and the gauge length 20 mm. Without releasing the sample from the grips, the deformed strip (i.e., the temporary shape) was fixed by quenching the sample to 0 °C with the aid of ice spray. Then the sample was removed from the dynamometer clamps, and immediately transferred to the second (recovery) step. Recovery was quantitatively assessed using a TMA analyzer in tensile mode and applying the following non-conventional procedure: the applied force was set to zero and the change of sample length was recorded during a heating scan at 2 °C/min from 0 to 70 °C. During this process, changes of stress (d), strain (e), and temperature (T) of the copolymer were recorded.
3. Results and discussion 2.5. Measurements 3.1. Synthesis and characterization The molecular weights of the polymers were determined on a Waters apparatus equipped with an RI detector. THF was used as the mobile phase at a flow rate of 1.0 ml/min. 20 ll of 1.0 w/v% solution was injected for each analysis. Calibration was accomplished with polystyrene standards (Polysciences, Warrington, PA). The copolymer composition was determined by 1H NMR spectroscopy. The 1H NMR spectra of the copolymers were recorded at a Bruker AV500 NMR spectrometer at ambient temperature, using DMSO as solvent, and TMS as the internal reference. The spectra were obtained with a FID resolution of 0.245 Hz/point, corresponding to a sweep width of 8 kHz, acquisition time was 2.04 s. Differential scanning calorimetry (DSC) was registered with a Perkin-Elmer DSC Pyris-6 instrument, at a heating rate of 10 °C/min. About 10 mg of product was used for each analysis. The glass transition temperature (Tg) was taken at the midpoint of the transition zone on the second heating scan. The surface of degraded samples of all the polymers was examined by using a Philips XL30 ESEM Tungsten Scanning Electron Microscope under reduced pressure (5 Torr). Stress–strain measurements were carried out on strips die-cut from solvent-cast sheets by means of an Instron 1121 tensile testing machine at room temperature. Strips with 0.5 mm thickness and 3 mm width were used. The cross-head speed was 40 mm/min, and the gauge length was 20 mm. Six measurements were performed for each polymer.
PTMC homopolymer and PTMC–PDLLA copolymers with TMC/DL-LA feed ratios of 2/1, 1/1 and 1/2 denoted as PTDLA21, PTDLA11 and PTDLA12 were synthesized by bulk ring-opening polymerization of 1,3-trimethylene carbonate and D,L-lactide. Zn(Lac)2 was used as catalyst instead of stannous octoate or other organometallic catalysts which are more or less cytotoxic. The 1H NMR spectrum of PTDLA11 copolymer is shown in Fig. 1. Signals in the 4.9–5.2 ppm zone and at 1.5 ppm are assigned to –CH– and –CH3 of lactyl units, and those at 4.2 and 2.0 ppm to –CH2 of TMC units, respectively [30]. Interestingly, signals in the 4.9–5.2 ppm zone can be divided in two groups: the downfield group around 5.2 ppm belongs to main chain lactyl units, and the upfield group around 5.0 ppm is assigned to lactyl units linking to TMC units. The presence of many TMC-linking lactyl units indicates the random nature of the chain structure. The composition of the copolymers is calculated from the integrations of the signal at 4.0–4.2 ppm for TMC units and that at 5.0– 5.3 ppm for DL-LA units. As shown in Table 1, the [TMC]/ [LA] molar ratio is 67/33 and 48/52 for PTDLA21 and PTDLA11, respectively, which is very close to the feed ratio. In contrast, the TMC content in PTDLA12 is lower than in the feed, probably due to the lower reactivity of TMC monomer as compared to lactide, as reported in literature [31]. GPC was used to evaluate the molecular weights and polydispersity of the polymers. The number average molecular weights (Mn) of PTMC, PTDLA11 and PTDLA12
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Fig. 1. 1H NMR spectrum of PTDLA11 copolymer in DMSO.
are all above 100,000, whereas the Mn of PTDLA21 is slightly lower (Table 1). On the other hand, the polydispersit index (Ip = Mw/Mn) is in the range of 1.7–1.9. Therefore, Zn(Lac)2 is an efficient catalyst for the copolymerization of TMC and LA monomers. The thermal properties of the polymers were determined by DSC. All the polymers are intrinsically amorphous as a single glass transition is detected, in agreement with the random chain structure. The Tg of PTMC is detected at 10.6 °C, indicating that it is a rubbery polymer at room temperature [32]. The Tg of PTDLA copolymers increases with increasing D,L-LA content (Table 1), in agreement with the fact the Tg of PDLLA (50 °C) is much higher than that of PTMC.
slowly as no mass loss is detected, in agreement with the literature [33]. In contrast, PTDLA copolymers appear much more hydrolytically degradable. No mass loss is observed for PTDLA21 and PTDLA11 during the first 9 weeks, while the mass loss of PTDLA12 reaches 3.4% probably due to the release of low molecular weight species such as residual monomers [34]. Mass loss slightly increases for PTDLA21 and PTDLA11 to reach 3.5% after 21 weeks. Beyond, mass loss rapidly increases for the three copolymers. The final mass loss is 49% for PTDLA21, 49% for PTDLA11 and 50% for PTDLA12 at week 53. Apparently, PTDLA copolymers degrade much faster than PTMC homopolymer since ester bonds are more susceptible to hydrolysis than carbonate bonds as previously observed [20]. The hydrolysis rate of polyesters is influenced by the access to water for the labile ester bonds, Water uptake occurs when a polymer sample is immersed in an aqueous medium even though the polymer is hydrophobic [33]. Fig. 2b presents the water uptake profiles of the polymers during hydrolytic degradation. PTMC homopolymer appears very hydrophobic with only 3% of water uptake after
3.2. Hydrolytic degradation The hydrolytic degradation of the polymers was performed in pH 7.4 PBS at 37 °C. Fig. 2a shows the mass loss profiles of the polymers as a function of degradation time up to 53 weeks. PTMC homopolymer degrades extremely
Table 1 composition, molecular weight, thermal and mechanical properties of PTLA copolymers.
a b c d
Samples
Feed ratio (TMC/LA)
Molar ratioa (TMC/LA)
Tg (°C)
PTMC PTDLA21 PTDLA11 PTDLA12
100/0 67/33 50/50 33/67
100/0 67/33 48/52 18/82
10.6 11.4 22.4 44
Calculated from 1H NMR. Tensile strength. Stain at break. Young’s modulus.
GPC Mn
DPI
114,000 88,000 104,000 119,000
1.9 1.8 1.8 1.7
db (MPa)
ebreakc (%)
Ed (MPa)
1.4 0.41 0.57 0.73
720 650 620 540
4.7 2.0 2.1 2.4
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Fig. 4. Changes of LA content in the PTDLA copolymers during hydrolytic degradation.
Fig. 2. (a) Mass loss and (b) water uptake of the copolymers during hydrolytic degradation. Three duplicate samples were used for each data point.
21 weeks. PTDLA21 and PTDLA11 present slightly higher water uptake which reaches 8% and 9%, respectively. In
Fig. 3. Molecular weight profiles of the polymers during hydrolytic degradation as measured by GPC.
contrast, nearly 27% of water uptake is obtained for PTDLA12 in the same period. Higher water uptake is observed for PTDLA copolymers as compared to PTMC because degradation of PTDLA leads to formation of hydrophilic OH and COOH endgroups. GPC was used to monitor changes of molecular weights of the polymers during degradation. Fig. 3 presents the normalized Mn changes as a function of degradation time. The Mn of PTMC homopolymer appears almost unchanged during the degradation period up to 53 weeks, in agreement with the fact that this polymer is not degradable by pure hydrolysis. In contrast, the copolymers degrade much faster than PTMC. Mn decrease is 66% after 16 weeks for PTDLA12, 57% for PTDLA11 and 38% for PTDLA21. Therefore, the higher the TMC content, the slower the Mn decrease, in agreement with the non-hydrodegradability of PTMC. Beyond, Mn changes tend to converge for three PTDLA copolymers. Compositional changes of the copolymers during degradation were obtained from 1H NMR (Fig. 4). The composition remains almost unchanged during the first 21 weeks, in agreement with the fact that no significant mass loss is detected. Beyond, LA content decreases from 33% to 22% for PTDLA21, from 51% to 43% for PTDLA11, and from 82% to 78% for PTDLA12. These findings indicate that LA units are preferentially degraded in amorphous PTDLA copolymers, as previously reported [28]. However, the compositional changes are less than what could be expected from the mass loss (50%), indicating that TMC units are degradable in PTDLA copolymers although PTMC homopolymer is not degradable. PTDLA12 exhibits the smallest LA content decrease since it initially contains the largest LA content (82%). Comparison of the Mn decrease, mass loss data and compositional changes allows to better understand the hydrolytic degradation behaviors of the copolymers. Hydrolytic cleavage of copolymer chains immediately starts after immersion in the PBS, leading to rapid Mn decrease. Nevertheless, significant mass loss is observed much later, i.e., beyond 21 weeks. In fact, the Mn of the copolymers decreases from nearly 20,000 to 6000 from
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Fig. 5. (a) Mass loss and (b) water uptake of the polymers during enzymatic degradation. Three duplicate samples were used for each data point.
21 to 35 weeks. Low Mn species are thus formed and released into the medium, which results in rapid mass loss. LA units are preferentially degraded along the copolymer chains, leading to compositional changes.
the LA content, the faster the degradation in the presence of proteinase K which cannot degrade PTMC homopolymer. Fig. 5b illustrates the water uptake curves during enzymatic degradation of the polymers. PTDLA12 with LA content of 82% presents a gradual increase in water uptake, up to 15.7% after 264 h, whereas the water uptake of PTMC, PTDLA21 and PTDLA11 is approximately 3%. These findings confirm the higher bulk hydrophilicity of PTDLA12, in agreement with the data from hydrolytic degradation. The changes of molecular weights and compositions are followed by using GPC and NMR. The composition of the copolymers remains almost unchanged during the degradation period. The only exception is PTDLA12 whose LA content slightly decreases from 82% to 78% after 216 h, in contrast to the hydrolytic degradation data. This finding could be attributed to the very important mass loss of PTDLA12 (nearly 90%) after 216 h enzymatic degradation. On the other hand, the Mn of the PTDLA copolymers slightly decreases, while that of PTMC remains almost the same during enzymatic degradation. It is well known that enzymatic degradation is a surface erosion process which hardly affects the bulk properties, but hydrolytic degradation still takes place in the bulk. Similar findings have been reported in the case of PCL–PLLA block or random copolymers during degradation by pseudomonas lipase [36,37]. ESEM measurements were performed to examine the surface morphology of the polymers during enzymatic degradation. No changes are detected after degradation for PTMC due to the absence of degradation. Similarly, PTDLA21 presents little changes after degradation in agreement with the small mass loss (11%). In contrast, PTDLA11 and PTDLA12 appear largely eroded in the presence of proteinase K. The original samples exhibit a smooth or slightly rugged surface as illustrated in Fig. 6a and d for PTDLA11 and PTDLA12, respectively. However, a highly porous structure is detected for PTDLA11 (Fig. 6b and c) degraded after 120 and 216 h, and for PTDLA12 (Fig. 6e and f) after 120 and 216 h. Similar findings were previously reported for PLA [13,38]. Therefore, PTDLA copolymers are degradable in the presence of proteinase K, in particular for those with high LA contents. 3.4. Mechanical properties
3.3. Enzymatic degradation Proteinase K is largely used for enzymatic degradation of PLA-based homo- and copolymers [13,14,35]. The enzyme-catalyzed degradation of PTDLA copolymers was carried out using proteinase K in pH 8.5 Tris buffer at 37 °C. PTMC homopolymer was taken as control. Fig. 5a shows the mass loss curves of PTMC and PTDLA copolymers during degradation. PTMC appears non-degradable in the presence of proteinase K as no significant mass loss is detected after 264 h. In contrast, the copolymers exhibit various degradation rates. Mass loss of PTDLA21 increases slowly but constantly to reach 11% after 264 h. PTDLA11 presents faster mass loss which reaches nearly 37% in the same period. The highest mass loss (91%) is obtained for PTDLA12 containing 82% of LA units. The difference should be ascribed to the compositions of copolymers. The higher
The mechanical properties of high molecular weight PTMC homopolymer and PTDLA copolymers were evaluated by using an Instron 1121 testing machine with a tensile rate of 40 mm/min at room temperature. The tensile strength (d), strain at break (ebreak) and Young’s modulus (E) are listed in Table 1. PTMC exhibits an ebreak value of 719%, in agreement with its elastomeric behavior. Copolymerization of TMC with D,L-LA results in decrease of the ebreak. The higher the LA content, the lower the ebreak. Nevertheless, the ebreak of the copolymers are all above 500%. The tensile strength and Young’s modulus of PTMC are 1.4 and 4.7 MPa, respectively. Copolymerization of TMC with D,L-LA results in decrease of tensile strength and Young’s modulus due to disordered chain structure. On the other hand, an increase of both parameters is observed with increasing LA content among the three PTDLA
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Fig. 6. ESEM micrographs of PTDLA11 (a–c) and PTDLA12 (d–f) copolymers after 0, 120 and 216 h enzymatic degradation.
Fig. 7. Tensile dynamic mechanical analysis (DMA) measurements for PTMC homopolymer and PTDLA copolymers.
copolymers. This can be assigned to the fact that poly(D,Llactide) homopolymer is a thermoplastic with much higher tensile strength and Young’s modulus than PTMC homopolymer. DMA was used to determine the storage modulus (E0 ) and tan d of all the polymers as a function of temperature (Fig. 7). PTDLA12 exhibits the highest storage modulus, nearly 3.5 GPa at 10 °C. The storage modulus slightly decreases with increasing temperature, and a strong decrease is observed in the range from 30 to 45 °C. On the other hand, the storage modulus decreases with increasing TMC content, PTMC homopolymer exhibiting the lowest E0 value. The tan d of the copolymers exhibits a peak value corresponding to the glass transition, which corroborates with data obtained from DSC analysis. PTDLA11 presents the highest tan d value of nearly 2.5, indicating its high energy storage and recovery ability. Moreover, the Tg of PTDLA11 is detected around the room temperature. PTDLA11 is thus selected for further investigations.
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Fig. 9. Shape memory experiment on a strip of PTDLA11 copolymer: the permanent shape (bend, a); the temporary shape (spiral, b); the recovery process at 37 °C approximately (c) after 2 s and (d) after 10 s.
3.5. Shape memory behavior of PTDLA11
Fig. 8. Stress–strain properties of the PTDLA11 copolymer upon cycle loading (a) cycles 1–20, and (b) cycles 21–23 (after 2 h relaxation).
The shape memory properties of the PTDLA11 copolymer with a Tg of 22.4 °C were evaluated using a hot pressed film between two selected temperature (37 and 0 °C). Fig. 9 shows the permanent shape, the temporary shape, and the recovered shape after heat treatment. Firstly, PTDLA11 (a bend strip, Fig. 9a) were stretched and wrapped around a rod in an incubator at 37 °C to reach an elongation higher than 150% (step 1), and then it was rapidly cooled down to 0 °C (step 2), the temporary shape (spiral, Fig. 9b) is obtained. The shape recovery process (step 3) is performed at 37 °C as shown in Fig. 10c and 10d. After 2 s and 10 s, the strip progressively recovers its permanent shape. Fig. 10 summarizes the results of a typical shape memory experiment in a three-dimensional graph. Path 1 from position A to position B is a stress–strain curve at a
Fig. 8 shows the typical stress–strain curves of PTDLA11 recorded for different loading cycles. PTDLA11 appears highly elastic. The residual strain is approximately 4% after the first cycle at a strain of 50%. Nearly 80% recovery is obtained after 20 cycles. The cycle loading tests show that the mechanical properties of the copolymer are affected by repeated loading, especially after the first and second cycles. The hysteresis shape becomes narrower during the following cycles. Every cycle loading test results in a mechanical hysteresis due to the viscoelastic nature of the polymer, while the difference between the ascending curves of two successive cycles can be explained by the non-reversible reorientation of macromolecular chains. After 20 cycles, a recovery period of 2 h was applied and 3 more cycles were recorded. At the beginning of the 21st cycle, PTDLA11 exhibits a permanent set of approximately 6%, i.e., much lower than the value of 20% obtained after 20 cycles. This finding can be assigned to relaxation of chains during the 2-h recovery period. After the 21st cycle, similar stress–strain curves are observed. Meanwhile, the residual strain in the 21st cycle is up to approximately 11%, showing that repeated loading and relaxation have an important effect on the structure of the linear polymer.
Fig. 10. Thermomechanical experiment on a strip of PTDLA11 copolymer: (1) stress–strain curve at 37 °C (strain rate, 20 mm/min); (2) recovery process (heating rate, 2 °C/min).
J. Yang et al. / European Polymer Journal 46 (2010) 783–791
constant temperature (37 °C) preformed in the dynamometer, the 150% strained strip is taken as the temporary shape and corresponds to the maximum strain (em). After quenching down to 0 °C and the load withdrawn, the sample can recover from position C to position D at a heating rate of 2 °C/min as path 2 performed in TMA. The recorded residual strain (er) of PTDLA11 after one cycle is 25%. Because the deformation recovery ability of the sample can be quantified by the shape recovery ratio parameter (Rr), defined as follows:
Rr ¼ ðem er Þ=em
ð3Þ
where em is the strain imposed to the strip at 37 °C and er is the measured residual strain. The Rr of PTDLA11 is found to be 83%. The initial recovery temperature taken from the intersection of tangent and baseline prior to recovery is 23 °C, i.e., very close to its Tg. It is commonly accepted that the polymer with shape memory behavior should possess two components at the molecular level: a fixing phase constituted by physical or chemical cross-links to fix the shape and a reversible phase to provide the elastomeric deformation. Thermo-responsive SMPs are generally networks. However, Zini et al. [6] reported the linear PLLA–PGA–PTMC terpolymers exhibit shape memory behavior by chain entanglements. It is also the case of linear PTDLA11 copolymer with high molecular weight. Time and temperature strongly influence the shape memory behavior of amorphous copolymers. It appears that temperature and deformation time must be kept as low as possible to restrain disentanglement of polymeric chains leading to irreversible flow. 4. Conclusions The hydrolytic and enzymatic degradation of PTMC homopolymer and PTDLA copolymers were investigated under in vitro conditions. PTMC homopolymer degrades extremely slowly by pure hydrolysis or in the presence of proteinase K. In contrast, PTDLA copolymers with different compositions degrade at various rates both in PBS and in enzyme solutions. The higher the LA content, the faster the degradation. LA units are preferentially degraded during hydrolytic degradation, indicating that ester bonds are more susceptible to hydrolytic cleavage than carbonate ones. Changes in surface morphology are observed during enzymatic degradation, in agreement with surface erosion process. The PTDLA11 copolymer with equivalent TMC/LA contents is highly elastic with outstanding shape memory property. Therefore, high molecular weight PTDLA copolymers are promising candidates for clinical applications in minimally invasive surgery. Acknowledgements The authors acknowledge the National Basic Research Program of China (973 Program No. 2007CB935801), the National Natural Science Foundation of China (No.
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