Polymers from Biocatalysis: Materials with a Broad Spectrum of

Chapter 14

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Polymers from Biocatalysis: Materials with a Broad Spectrum of Physical Properties Mariastella Scandola,a,* Maria Letizia Focarete,a and Richard A. Grossb aDepartment

of Chemistry ‘G. Ciamician’, University of Bologna, Via Selmi 2, 40126 Bologna (Italy) bNSF-I/UCRC Center for Biocatalysis and Bioprocessing of Macromolecules, Department of Chemical and Biological Sciences, Polytechnic University, Six Metrotech Center, Brooklyn, New York 11201 *[email protected]

Copolymers of ω-pentadecalactone (PDL) with ε-caprolactone, valerolactone, dioxanone and trimethylenecarbonate synthesized by biocatalysis show rather uncommon crystallization behavior, namely cocrystallization of the monomer units that leads to highly crystalline copolymers over the whole composition range. Hydrophilic/hydrophobic balance can be adjusted by a suitable choice of the comonomer and of composition, leading to materials with tunable hydrolytic degradation rate for environmental and biomedical applications. Copolyestercarbonates, copolyesteramides and polyol-containing copolyesters synthesized by lipase-catalysed polycondensation show strongly composition dependent physical properties, that can be easily tailored by composition control and cover the whole range from hard solid materials down to gluelike substances.

Introduction High molecular weight polymers that cannot be obtained by chemical routes are easily synthesized by lipase-catalyzed polymerization (1). Some lipases such as Candida antarctica Lipase B (CALB), when used in ring opening polymerization or polycondensation reactions, allow incorporation of different © 2010 American Chemical Society In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


monomers along chains so that copolymers with defined composition and microstructure can be prepared. A number of such copolymers, i.e. copolyesters (2–7), copolyestercarbonates (8–10) and copolyesteramides (11), have been successfully synthesized and their solid-state properties have been characterized. Copolymers show a wide range of thermomechanical properties that can be finely tuned through composition and microstructure control, which in turn can be tailored by playing on feed ratio and reaction conditions. Such a control is critical to the ultimate goal of adjusting physical, mechanical, and biological properties of the copolymers in view of their materials applications. Metal-free catalysis, mild reaction conditions, presence of hydrolysable bonds along the chains, tunable hydrophilic/hydrophobic ratio suggest potential uses of these polymers as bioresorbable materials in the medical field (surgery, implants, drug delivery, etc). Additional interesting applications of the biosynthesized polymers regard the area of environmentally friendly biodegradable materials.

Experimental Materials All polymers were synthesized using Candida antarctica Lipase B (CALB, Novozyme-435) as described elsewhere (2, 4–6, 8, 10–12). Solvents used for electrospinning (chloroform, dichloromethane, 1,1,1,3,3,3-Hexafluoro-2propanol) were Aldrich products used without further purification. Instrumentation Polymer solid-state properties were characterized by differential scanning calorimetry (TA DSC-Q100, equipped with LNCS low-temperature accessory), thermogravimetric analysis (TA Instruments TGA2950), dynamical mechanical analysis (DMTA, Polymer Labs., MKII), wide angle X-ray diffraction (WAXS, PANalytical X’Pert PRO), tensile testing (INSTRON 4465) and scanning electron microscopy (SEM, Philips 515). The apparatus used for electrospinning was made in house (13) as was the system employed for the production of scaffolds through supercritical carbon dioxide (sc-CO2) foaming (14).

Results and Discussion Poly(ω-pentadecalactone) Poly(ω-pentadecalactone), PPDL, synthesized by biocatalysis is a high molecular weight (>100 KDa) polyester that crystallizes from the melt with very fast kinetics. It is highly crystalline and melts around 100°C. It shows a pseudo-orthorombic monoclinic unit cell with dimensions a=7.49(1), b=5.034(9), and c=20.00(4)Å (fiber axis), and α= 90.06(4)°, that hosts two monomeric units belonging to polymer chains with opposite orientation (15). The glass transition of this highly crystalline polymer, that is barely detectable by DSC, was determined by DMTA and lays around -30°C (12). 202 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


The mechanical properties of PPDL resemble those of low density polyethylene, LDPE (Figure 1). Both polymers exhibit a hard and tough behavior, the curve of PPDL showing a steeper slope in the initial linear part, i.e. higher elastic modulus, as well as higher stress at yield than LDPE. Good mechanical properties associated with the presence along the polymer chain of hydrolysable ester linkages render PPDL an interesting biodegradable material for diversified purposes, including biomedical applications. In this context PPDL is a good candidate as a bioresorbable material where long healing times are required, thanks to the long methylene sequence in the monomer unit (14 C atoms) that ‘dilute’ the hydrolizable ester bonds along the chain. PPDL scaffolds for tissue engineering were fabricated by means of electrospinning technology. Biocompatibility of PPDL and ability of the scaffolds to support cell growth were demonstrated using embryonic rat cardiac H9c2 cells (13). Figure 2 shows SEM micrographs of an electrospun nonwoven mat of PPDL fibers (average diameter 600 nm) before and after 14 days of culture. The seeded cells are seen to propagate and spread over the PPDL mat surface while retaining their native morphology. Cytotoxicity tests confirmed biocompatibility of PPDL (13). Highly Crystalline ω-Pentadecalactone Copolymers Random copolyesters of pentadecalactone (PDL) were synthesized using as co-units caprolactone (CL), valerolactone (VL) and dioxanone (DO) over the whole range of molar compositions (2, 4). The solid-state behaviour of such copolymers is very peculiar because, despite the fact that the two monomers are randomly distributed along the chain, high crystallinity develops at all compositions (3, 4). However, while both poly(PDL-co-CL) and poly(PDL-co-VL) are isomorphic systems (3), i.e. they crystallize in a lattice that smoothly changes with copolymer composition from that of one homopolymer (PPDL) to that of the other (PCL or PVL), poly(PDL-co-DO) shows isodimorphic behavior (4). The latter case occurs when the crystal lattice of each homopolymer is able to host foreign comonomer units only up to a certain degree. As a consequence, two crystal phases develop: PPDL-type in PDL-rich copolymers and poly(dioxanone)-type at the other end of the composition range. In isodimorphic systems the melting temperature changes with composition and shows a minimum at the so-called pseudo-eutectic, where the two crystal phases may be found to coexist. In poly(PDL-co-DO) the pseudo-eutectic lays at 71mol% DO content, a composition where both PPDL-type and PDO-type crystals are revealed by WAXS (4). Figure 3 compares the melting behavior of copolymers of PDL with CL and with DO, i.e. of systems that show isomorphic and isodimorphic behavior respectively. An unusual crystallization behavior, involving cocrystallization, was also observed in chemo-enzymatically synthesized methacrylate brush copolymers carrying a PPDL block linked through a PEG spacer to the side chain (16). It was found that in these methacrylate polymer brushes a PPDL-type crystal phase develops upon side-chain crystallization. WAXS analysis revealed that, unexpectedly, the PEG segments were incorporated into the PPDL crystal lattice 203 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


Figure 1. Stress-strain curves of PPDL and LDPE films (room temperature; strain rate: 10 mm/min)

Figure 2. Left: Scanning electron micrograph of an electrospun PPDL fiber mat; right: PPDL fiber mat with H9c2 cells after 14 days of culture. in the unusual extended zigzag conformation (PEG normally adopts a 72 helical conformation). This result further demonstrates the very peculiar ability of the long PDL unit to induce different moieties to enter the PPDL lattice via cocrystallization. In all systems investigated it was found that copolymerization of smaller lactones with PDL is an effective way to enhance thermal stability of the corresponding homopolymers. This is illustrated in Figure 4 for a poly(PDL-co-VL) and a poly(PDL-co-DO) with near-equimolar PDL and VL content. Given the high hydrophobicity of the PDL unit, copolymerization with more hydrophilic smaller lactones is also a means to tailor hydrolytic degradation rate of the resulting copolymers. Table 1 collects the results of water contact angle measurements on compression molded films of PPDL and of copolyesters with similar PDL molar content. Substitution of PDL units with CL or VL moieties decreases the contact angle, more markedly in the case of the shorter (VL) unit, as expected. Comparison of the results for the two five-chain-atom monomer units, VL and DO, highlights the effect of the presence of the oxygen atom in the DO repeat that additionally lowers contact angle. Copolymerization is 204 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


Figure 3. Composition dependence of melting temperature of: (▲) poly(PDL-co-DO), isodimorphic system, and (○) poly(PDL-co-CL), isomorphic system.

Figure 4. Comparison of TGA curves of homopolymers PVL, PDO and PPDL with those of poly(PDL-co-50mol%VL) and poly(PDL-co-57mol%DO) therefore a simple way to manipulate hydrophilicity and, consequently, hydrolytic degradation rate in view of potential biomedical applications of these polyesters. The mechanical properties of PDL-copolymers reflect the presence of a rigid crystal phase associated with a mobile amorphous phase (all copolymers of PDL with CL, VL or DO have glass transition temperature, Tg, lower than room temperature). The amount of crystal phase, that depends on comonomer type and content, influences the stress-strain behavior. As an example, Figure 5 compares the stress-strain curves of two copolymers - poly(PDL-co-36mol%CL) and poly(PDL-co-53mol%DO) – with that of PPDL. The three curves are remarkably different, mainly in terms of elastic modulus and strength. Table 2 reports tensile modulus and melting enthalpy (as an indication of crystallinity degree). Both parameters decrease in the same order, i.e. PPDL > poly(PDL-co-36mol%CL) > poly(PDL-co-53mol%DO), showing that the observed differences in mechanical properties are maily associated with different crystal phase content in the samples. Knowledge of structure-property 205 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


Table 1. Water Contact Angle of PPDL and of Copolyesters sample

PDL (%mol)

PDL (%wt)

Contact Angle (°)

SD

PPDL

100

100

106

3

poly(PDL-co-CL)

50

79

95

2

poly(PDL-co-VL)

45

66

87

2

poly(PDL-co-DO)

47

68

76

3

relations in these copolymers (2–4, 7) teaches that crystallinity degree can be changed to a certain extent by playing on composition. Thus the material’s properties can be adjusted to specific needs. In summary, PDL copolymers can be biosynthesized with tunable crystallinity degree, hydrophilicity and mechanical properties suitable for a wide variety of applications. Among others, an important biomedical field of activity regards the use of scaffolds for cell expansion in tissue engineering. Scaffolds of PDL copolymers were therefore produced not only via electrospinning technology (13) as mentioned above, but also through sc-CO2 foaming (14). Figure 6 shows, as an example, the 3-D reconstruction by micro computer tomography (µ-CT) of a foamed porous scaffold of poly(PDL-co-36mol%CL). In order to optimize foaming of this highly crystalline material, a novel experimental procedure was setup that included controlled cooling during the depressurization stage (14). By this new method scaffolds with tunable porosity, pore size and interconnectivity were successfully obtained with mechanical properties suitable for applications in cartilage tissue regeneration. A copolyester-carbonate containing the PDL unit was also synthesized using CALB (8). The peculiar crystallizing ability of PDL and its tendency, when copolymerized with the cyclic trimethylenecarbonate monomer (TMC), to yield alternate comonomer sequences led to highly crystalline poly(PDL-TMC) copolymers. Worth noting is that the homopolymer poly(TMC) is totally amorphous. Nevertheless a new crystal phase, that melts at lower temperature than PPDL and is attributed to crystallization of PDL-TMC alternate sequences, was observed (9). Moreover the copolymers showed enhanced thermal stability compared with that of PTMC, as illustrated for the copolymer with 50mol%TMC in Figure 7.

Copolyestercarbonates, Copolyesteramides and Polyol-Containing Copolyesters with Strongly Composition Dependent Physical Properties Poly(butylene carbonate-co-butylene succinate) random copolymers were synthesized by lipase catalysis over the whole range of comonomer content. Poly(BC-co-BS) copolymers varied from semicrystalline to near completely amorphous with changing composition (10). The semicrystalline poly(BC-co-BS) show only one crystal phase, either that of the homopolymer poly(BC) or that of poly(BS), indicating the inability of either crystal lattice to host different 206 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


Figure 5. Comparison of stress-strain curves of poly(PDL-co-36mol%CL), poly(PDL-co-53mol%DO) and PPDL films (room temperature; strain rate: 10 mm/min) Table 2. Mechanical properties of PPDL and of selected copolymers (strain rate: 10 mm/min)

a

sample

Tensile modulus (MPa)

Melting enthalpy a (J/g)

PPDL

530 ± 43

125

poly(PDL-co-36mol%CL)

230 ± 17

101

poly(PDL-co-53mol%DO)

85 ± 2

58

From DSC

Figure 6. Three-dimensional reconstruction by µ-CT of a porous scaffold of poly(PDL-co-36mol%CL) obtained by sc-CO2 foaming. Scale bar: 1 µm. comonomer units. At particular copolymer compositions (around 70 mol % BC), neither BC units nor BS units are able to organize into a crystal structure. As a consequence of the disappearance of crystallinity the material, characterized by a low Tg (around -40°C irrespective of composition), changes from rigid to soft 207 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


Figure 7. TGA curves of PTMC, PPDL and Poly(PDL-50mol%TMC)

Figure 8. Composition dependence of melting enthalpy in copolymers of octamethylene adipate with: (4) glycerol adipate, (▲) sorbitol adipate and (●) silicone adipamide. and sticky. It is reasonable to expect that the susceptibility to hydrolysis of these copolyestercarbonates will parallel the decrease of crystallinity. Polyol-containing polyesters were also syhthesized using CALB (5, 6) and they were found to show a similar decrease of crystallinity with increasing amount of comonomer units (7). Figure 8 shows the decrease of melting enthalpy that is displayed by copolymers of octanediol adipate with either sorbitol adipate or glycerol adipate with increasing polyol content. Worth mentioning is that, concurrent with the decrease of crystallinity, the increasing amount of hydrophilic 208 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


Figure 9. Left: hard crystalline P(OA-co-10mol%SiAA); right: gluelike P(OA-co-50mol%SiAA) polyol units in the polyester chain may enable fine tuning of the degradation rate of these copolyesters. Moreover, the hydroxyl functional groups on the polyol moieties along the chain may be exploited to link biomolecules, making this class of polymers attractive candidates for use as bioactive and bioresorbable materials. CALB was also used to catalyse one-pot reactions between diethyl adipate (DEA), 1,8-octanediol (OD), and α,ω-(diaminopropyl)polydimethylsiloxane (SiNH2) under mild conditions (11). The obtained silicone polyesteramides, that exhibit blocklike sequence distribution, show physical properties that strongly depend on the relative amount of amide and ester units along the polymer chain. High content of DEA-OA units leads to hard solid materials containing a well developed high-melting poly(octamethylene adipate)-type (POA) crystal phase, whose melting temperature and enthalpy change with composition. The decrease of melting enthalpy (i.e. of crystallinity) with increasing silicone adipamide content is shown in Figure 8, where comonomer content is reported in terms of weight fraction. When the DEA-SiAA units mole content is ≥ 33% (66wt% in Figure 8), the material acquires a sticky appearance. As an example, Figure 9 compares the picture of hard crystalline P(OA-co-10mol%SiAA) with that of gluelike P(OA-co-50mol%SiAA), with the aim to illustrate the wide range of physical properties that these materials can display.

Conclusions The copolymers of PDL, synthesized by biocatalysis, show a peculiar and rather uncommon crystallizing behavior, namely cocrystallization of the comonomer units leading to either isomorphic substitution or to isodimorphism. This behavior is associated with the peculiar crystallizing ability of the long PDL unit, that is able to incorporate into its polyethylene-type crystal lattice a number of different monomer units. These crystalline copolymers with tunable hydrolytic degradation rates can be used in medical applications, for example as scaffolds for tissue engineering. Such cell supporting structures have been 209 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

produced using PPDL and PDL-copolymers both by electrospinning technology and by supercritical CO2 foaming. On the other hand, biosynthesized condensation copolymers, whose amount of crystal phase decreases with increasing comonomer content, display solid state properties that cover the whole range from hard solid materials down to gluelike substances. Such properties can be easily tailored by composition control and can be adjusted to meet the requirements of a broad range of applications.


Acknowledgments Financial support from Italian Ministry of Foreign Affairs (Directorate General for Cultural Promotion and Cooperation - Significant Bilateral Project Italy-USA) is gratefully acknowledged.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

14. 15.

Gross, R. A.; Kumar, A.; Kalra, B. Chem. Rev. 2001, 101, 2097–2124. Kumar, A.; Kalra, B.; Dekhterman, A.; Gross, R. A. Macromolecules 2000, 33, 6303–6309. Ceccorulli, G.; Scandola, M.; Kumar, A.; Kalra, B.; Gross, R. A. Biomacromolecules 2005, 6, 902–907. Jiang, Z.; Azim, H.; Gross, R. A.; Focarete, M.L.; Scandola, M. Biomacromolecules 2007, 8, 2262–2269. Kulshrestha, A.; Kumar, A.; Gao, W.; Gross, R. A. Polym. Prepr., Am. Chem. Soc. 2003, 44, 585–586. Kumar, A.; Kulshrestha, A.; Gao, W.; Gross, R. A. Macromolecules 2003, 36, 8219–8221. Fu, H.; Kulshrestha, A.S.; Gao, W.; Gross, R. A.; Baiardo, M.; Scandola, M. Macromolecules 2003, 36, 9804–9808. Kumar, A.; Garg, K.; Gross, R. A. Macromolecules 2001, 34, 3527–3533. Focarete, M.L.; Gazzano, M.; Scandola, M.; Gross, R. A. Macromolecules 2002, 35, 8066–8071. Zini, E.; Scandola, M.; Jiang, Z.; Liu, C.; Gross, R. A. Macromolecules 2008, 41, 4681–4687. Sharma, B.; Azim, A.; Azim, H.; Gross, R. A.; Zini, E.; Focarete, M. L.; Scandola, M. Macromolecules 2007, 40, 7919–7927. Focarete, M. L.; Scandola, M.; Kumar, A.; Gross, R. A. J. Polym. Sci.: Part B, Polym. Phys. 2001, 39, 1721–1729. Focarete, M. L.; Gualandi, C.; Scandola, M.; Govoni, M.; Giordano, E.; Foroni, L.; Valente, S.; Pasquinelli, G.; Gao, W.; Gross, R. A. J. Biomater. Sci. (Polymer Ed.) 2010, in press. Gualandi, C.; White, L. J.; Chen, L.; Gross, R. A.; Shakesheff, K. M.; Howdle, S. M.; Scandola, M. Acta Biomater. 2010, 6, 130–136. Gazzano, M.; Malta, V.; Focarete, M. L.; Scandola, M.; Gross, R. A. J. Polym. Sci.: Part B, Polym. Phys. 2003, 41, 1009–1013. 210 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


16. Kalra, B.; Kumar, A.; Gross, R. A.; Baiardo, M.; Scandola, M. Macromolecules 2004, 37, 1243–1250.

211 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

Polymers from Biocatalysis: Materials with a Broad Spectrum of

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