PLA-PEO-PLA Hydrogels: Chemical Structure, Self-Assembly and

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Chapter 18

PLA-PEO-PLA Hydrogels: Chemical Structure, Self-Assembly and Mechanical Properties Surita R. Bhatia1,* and Gregory N. Tew2,* 1Department

of Chemical Engineering, University of Massachusetts Amherst, Amherst, MA 01003 2Department of Polymer Science and Engineering, University of Massachusetts Amherst, Amherst, MA 01003 *Corresponding Authors. Email: [email protected], [email protected]

Poly(lactide) – block – poly(ethylene oxide) – block – poly(lactide) [PLA-PEO-PLA] triblock copolymers are known to form physical hydrogels in water. Their biodegradability makes them attractive for soft tissue scaffolds and drug delivery applications. In addition, they serve as an excellent model system for physical hydrogels or networks. Understanding their mechanical properties is an important objective. More recently, these non-covalent networks were converted to covalently cross-linked hydrogels that prevent the gel-to-sol transition upon exposure to infinite water. Specifically, this was accomplished by allowing the system to self-assemble followed by covalent capture of the self-assembled architecture.

Introduction Polyesters remain an important class of materials for biomaterials applications from small area tissue repair to the growth of new organs (1–4). Their overwhelmingly attractive features are their biodegradability and ease of synthesis; however, with respect to the optimal requirements of a scaffold, these polymers are far from ideal because they are very hydrophobic. Although PLA-based materials are important technologically, they suffer from unwanted responses in vivo including foreign body response (5). At the same time, the © 2012 American Chemical Society In Degradable Polymers and Materials: Principles and Practice (2nd Edition); Khemani, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


modulus of homopolyesters is quite far from many biological tissues. Although PLA and other synthetic polyesters, like poly(caprolactone), represent important materials for medical implants, they all suffer from a variety of critical limitations.

One approach to enhancing the water-solubility of these polyesters is to make block copolymers containing a water soluble block such as polyethylene oxide (PEO) (6). Here the discussion is confined to copolymers composed of biodegradable hydrophobic poly(lactic acid) (PLA), including the two stereoisomers L and D, and biocompatible hydrophilic poly(ethylene oxide) (PEO). Both AB and ABA block copolymer architectures have been studied in some detail (6–30). These architectures can be used to create physically cross-linked hydrogels with transitions near body temperature. Beyond the control of block lengths, the ability to control D,L stereochemistry directly impacts hydrogel modulus through crystallinity of the PLA blocks since pure L or D polymers are semi-crystalline but mixtures of D and L produce amorphous materials (12, 19, 21, 29, 31, 32). It has been observed that gel-forming PLA-PEG diblock systems can be effectively modulated to vary a number of important physical properties including permeability and degradation rate. The structural properties of the gel matrix (micromorphology and pore size) are directly related to the mass transport of water into the gel and transport of drug out of the polymer. As a result, the chemical composition and microstructure design can produce tailor made polymer matrices. As a class of materials, PLA-PEO-PLA materials are extremely versatile given their relatively simple chemical structures. They represent an important sub-division of the much broader area of hydrogels. Hydrogels remain of great interest in the area of biomaterials (15, 29, 30, 33–38). There self-assembly is driven by the association of the hydrophobic endblocks into micelles, which are bridged by the water-soluble midblocks forming physically crosslinked networks. These physical hydrogels are attractive because no crosslinking agent is necessary and the gelation can be triggered by concentration, temperatue, pH, salt, etc. At the same time, the use of chemical cross-links have also been studied (30, 39–41). Chemical crosslinking leads to a more permanent three-dimensional structure than the physically crosslinked counter-parts, but can still be degraded with time. A variety of crosslinking techniques, polymer structures, and architectures have been used to synthesize biodegradable hydrogels; however, the corresponding mechanical properties are not as well characterized. Since the 314 In Degradable Polymers and Materials: Principles and Practice (2nd Edition); Khemani, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


overall mechanical environment affects cell function, this is unfortunate (33, 42, 43). Cells typically bind to the extracellular matrix via surface receptors to generate traction forces. Cells can sense the restraining force of the substrate and respond by strengthening its cytoskeleton (44). Since the healthy survival of cells depends on the mechanical properties, it is important to consider these properties. Mechanical properties impact all types of processes including cellular structure, metabolism, transcription/translation, and even viability (44–50). Pioneering work showed NIH3T3 fibroblasts underwent compliance dependent motility changes (49, 51). Similar studies examined endothelial cells, myocytes, hepatocytes, neural/glial cells, and chondrocytes (52–56). Glial cells were unable to survive in soft materials, unlike neurons (52, 54, 57, 58). The use of hydrogels has been proposed to limit scar tissue as a result of mismatch of mechanical properties at the wound site (52). Motivated by these and other factors, we have studied PLA-PEO-PLA triblock copolymers over the last decade. Through these studies some insight into the structure-property relationships of PLA-PEO-PLA gels was obtained. Factors like dependency of gel strength on the molecular composition and polymer concentration, which directly impact gel structure, were investigated. This includes varying the stiffness by manipulating the length of the PLA endblocks (6), by changing the physical crosslinks from amorphous to crystalline (32), by varying the synthetic technique (59), by incorporating nanoparticles (60), and by covalently capturing the self-assembled structure (26). This last approach is particularly intriguing since it combines the advantages of self-assembly with the stability of covalent crosslinks. Coupling self-assembly to hydrogel mechanical properties in novel phase-separated systems has provided new insight and properties (39).

Materials and Methods Materials L-lactide, D-lactide, and rac-lactide (Aldrich) were purified by recrystallization in dry ethyl acetate and by sublimation prior to polymerization. The α,ω-dihydroxy polyethylene glycol macro initiator with molecular weight 8,900 (PEG 8KDa, Aldrich) was dried at room temperature under vacuum prior to polymerization. Stannous (II) 2-ethyl hexanoate (Alfa Aesar) was used without further purification. Typical Synthesis of PLLA-PEO-PLLA Triblock Copolymer PLLA-PEO-PLLA triblock copolymers were synthesized by bulk polymerization. PEG was introduced into a dried polymerization tube. The tube was purged with nitrogen, and placed in an oil bath at 150 °C. Stannous (II) 2-ethylhexanoate was introduced under nitrogen to the molten PEG and stirred for 315 In Degradable Polymers and Materials: Principles and Practice (2nd Edition); Khemani, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

10 minutes followed by addition of L-lactide to the macroinitiator/catalyst melt. The polymerization was carried out at 150 °C for 24 h with stirring. after which it was quenched by methanol. The product was dissolved in tetrahydrofuran and precipitated in n-hexane. The process of dissolution/reprecipitation was carried out three more times. The copolymer was dried under vacuum at room temperature for two days.


Sample Preparation and Instrumentation The copolymers polydispersity are measured versus polyethylene oxide standards using GPC (HP 1050 series, a HP 1047A differential refractometer, and three PLgel columns (5 μm 50Å, two 5 μm MIXED-D) in dimethylformamide as eluting solvent at a rate of 0.5 mL/min rate at room temperature. The copolymer compositions are determined by 1H NMR (Bruker, DPX300, 300MHz spectrometer, d-chloroform). Gels were prepared by slow addition of dried polymer sample to a fixed volume of DI water (15 mL) followed by stirring and heating. Gels were then transferred to a Bohlin CVO rheometer for oscillatory measurements. A cone-and-plate geometry with a 4° cone, 40 mm diameter plate, and 150 mm gap was used for all experiments on hydrogels. For liquid samples with a low viscosity, a couette geometry was used. Stress amplitude sweeps were performed to ensure that subsequent data was collected in the linear viscoelastic regime. Frequency sweeps were performed at a constant stress (0.1 - 2.0 Pa, depending on the sample) in the frequency range 0.01 - 100 Hz. At high frequencies, a resonant frequency of the rheometer motor was observed; thus, data are reported up to a frequency of approximately 10 Hz, again depending on the particular sample. Crosslinking Chemistry PLA-PEO-PLA triblock copolymer (10.0 g, 0.760 mmol, 1 equiv) was weighed into a dried round-bottom flask, dissolved in toluene, and attached to a Dean-Stark trap with a condenser. The system was evacuated and purged with nitrogen 3 times. The condenser was turned on and the solution was stirred and refluxed to azeotropically distill the solution. The distilled solution was cooled to room temperature and then placed in an ice bath. Triethylamine (1.06 mL, 7.60 mmol, 10 equiv) was added dropwise, followed by dropwise addition of acryloyl chloride (0.617 mL, 7.60 mmol, 10 equiv), and stirred overnight. Triethyl amine/hydrochloric acid salt was removed by filtration over filter paper, and the toluene was evaporated. The product was taken up in THF, passed through a plug of basic alumina, and precipitated in hexanes. End-functionalized PLA-PEO-PLA (187 mg) was weighed into the wells of a 48-well cell culture plate. The plate was heated to 80°C in a vacuum oven for 1.5 hour to melt a polymer film. After heating, the plate was cooled to room temperature. A 0.05% w/v I2959 solution was prepared by weighing 26 mg I2959 in a vial, adding 52 mL of phosphate buffered solution, and heating and sonicating to dissolve. For a 25% w/v hydrogel, 0.745 mL of 0.05% w/v I2959 solution was added to each well plate and allowed to swell into a physical gel over 3 - 4 days. 316 In Degradable Polymers and Materials: Principles and Practice (2nd Edition); Khemani, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


After full swelling of the physical hydrogel, the well plates were irradiated with long-wave UV radiation (~ 365 nm) for 5 minutes, flipped upside-down (hydrogel thickness is approximately 8 mm), and irradiated for 5 more minutes to initiate the photocrosslinking. The hydrogel concentration was varied (10, 15, 25, 35, and 45% w/v) by adjusting the amount of dry polymer added to the well, while maintaining a constant volume of added photoinitiator solution.

Results and Discussion PLA-PEO-PLA Triblock Copolymers Synthesis Lactide has two stereoisomers, D-lactide and L-lactide. The resulting polymers, poly(D-lactide) (PDLA) and poly(L-lactide) (PLLA), are isotactic and semicrystalline. In contrast, polymers formed by atactic poly(D,L-lactide), or racemic poly(R-lactide) (PRLA), are amorphous (32). In a unique, and perhaps the simplest system, this allowed direct control over the crystallinity of the self-assembled hydrophobic micelle formed by these copolymers. Generally, triblock copolymers synthesized through bulk-synthesis are more asymmetric, which reduced the mechanical stiffness of the gel phase. This had a larger impact on amorphous PRLA copolymers than their crystalline counterpart (59). Although a number of copolymers has been prepared, the PEO block length is held constant and the degree of polymerization (DP) for PLLA is varied (typically from ~52 to ~72) (6, 32, 61–63). In all cases, the polymerization was stopped before complete monomer consumption since this leads to broader molecular weight distributions (MWD).

Mechanical Properties of These Hydrogels Mechanical properties of physically crosslinked hydrogels are typically characterized using shear rheometry, by exposing the material to an oscillatory shear stress at various frequencies. This leads to a determination of the storage modulus (G′ and the loss modulus (G′′), providing insight into the elastic and viscous responses of the material, respectively. Chemically crosslinked hydrogels are more typically characterized by measuring the stress in the material as strain is applied in compression. Implementing Hooke’s Law (σ = Eε, where σ is stress, ε is strain, and E is the Young’s or elastic modulus) to determine the slope of the stress-strain curve in the linear region at low strains yields the elastic modulus. However, Hooke’s Law only applies to linearly elastic materials, while hydrogels typically have non-linear stress-strain responses in compression. Other models are available including the modified Neo-Hookean model (64–66) or a model defined by Mooney (67) and Rivlin (68). Regardless, many researchers only apply Hooke’s Law despite the non-linear behavior of their materials. 317 In Degradable Polymers and Materials: Principles and Practice (2nd Edition); Khemani, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 1. PLA-PEO-PLA was modified with acrylate end-endgroups. Hydrophobic PLA is illustrated in green; hydrophilic PEO is in blue. This triblock copolymer forms physical crosslinks which are captured covalently by photocrosslinking the junction points to make them permanent.

Figure 2. Typical Stress versus Strain Curve in Compression. 25% w/v photocrosslinked PLA-PEO-PLA before degradation. Stress curve is non-linear and typical of soft rubbery materials. (inset) Schematic representation of the self-assembly followed by the photochemical cross-linking reaction as well as a photograph of the slightly oqaque hydrogel in excess water. This demonstrates its lack of a gel-sol transition upon dissolution.

Addition of water to these ABA triblock copolymers produces hard, physically associated gels typically above 16 wt%. The length of the PLA block directly impacts the wt% of this sol-gel transition. The longer the PLA block (up to a solubility limit), the lower the transition (6, 62). To better quantify the mechanical properties of these hydrogels, dynamic mechanical shear rheology 318 In Degradable Polymers and Materials: Principles and Practice (2nd Edition); Khemani, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


was employed. A plot of G′ vs. frequency for two polymers showed the gel with shorter PLA materials (52 PLA units, 20 wt %) forms a weaker gel than the one with the longer PLA blocks (72 PLA units,16 wt %). Stiffness increased by more than two decades, from ~100 Pa to 10,000 Pa, as the length of the hydrophobic block is increased by 20 units. Other PEO-containing systems with alkyl hydrophobe end-caps showed qualitatively similar trends (24). The elastic moduli of fluoroalkyl-capped PEO gels were insensitive to hydrophobe length at high frequency (69, 70). The elastic modulus >10 kPa provides materials that are almost an order of magnitude stiffer than previously reported polymers composed of similar chemistry (71, 72). The self-assembled network structure of these ABA triblock copolymers is dynamic. As more water is added, the distance between micelles increases, lowering the density of junction points. However, by introducing a photocrosslinkable moiety (see Figure 1), the self-assembled structure can be covalently captured (26). This method allows the differences between chemical and physical crosslinking in the same polymer hydrogel system to be studied. It also represents a new strategy to form hydrogels and is likely to lead to novel properties since it has the advantages of self-assembly and covalent, permanent crosslinks. In order to covalently capture the assembled hydrogel, the PLA-PEO-PLA triblock copolymer endgroups were functionalized with acrylates so that the self-assembled structure could be locked in by initiating photocrosslinking with ultra-violet radiation. The photocrosslinked PLA-PEO-PLA hydrogels remained intact when swollen in phosphate buffered saline solution (pH = 7.4) at body temperature (37 °C) for extended periods of time. This is in contrast to the physical hydrogels. Compression mechanical studies were performed to evaluate these photocrosslinked hydrogels. The Neo-Hookean constitutive relationship for rubbers was utilized to model the compression curves. In this model, the specific form of the strain energy function (U) depends on the first invariant of the deformation tensor (I1) by the constant, C1:

Where λi is equal to the extension ratio in the i-principal direction, or more specifically, the length in the i-direction over the initial (pre-stressed) length in the i-direction. The extension ratio is related to the strain, ε, by the following expression: λ = ε + 1. For the case of uniaxial compression and assuming the material to be incompressible:

319 In Degradable Polymers and Materials: Principles and Practice (2nd Edition); Khemani, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


By substituting into the strain energy expression and differentiating with respect to the extension ratio, an expression for stress (σ) is derived:

where the single parameter C1 is defined as half of the shear modulus, G (C1 = G/2). The same relationship can be derived using a statistical thermodynamic approach in which the distribution of end-to-end distances between crosslinks is assumed to be Gaussian (64–66). A typical stress-strain curve is shown in Figure 2 along with its fit using this Neo-Hookean model. The fit agrees well with the data. It predicts the observed non-linear behavior, suggesting a Gaussian distribution of chains and few entanglements or looping chains. Initially, the 25% w/v hydrogels had a shear modulus of ~ 64 kPa. As they degraded, the shear modulus decreased exponentially to a value of ~ 7 kPa over 35 days. This agreed well with the swelling data.

Structural Properties of the Gel Phase The structure of these hydrogels containing crystalline PLLA domains was investigated by WAXS, ultra small angle x-ray scattering (USAXS), ultra small angle neutron scattering (USANS) and confocal microscopy techniques (6, 31, 60–63). It was expected that the structure of a hydrogel would strongly influence its mechanical properties which are known to be dependent on both the nanoscale and microscale structure (61, 73). All gel samples exhibited low q range scattering that followed power law behavior (61), indicating scattering from fractal structures (74, 75). The power law exponents were found to be less than or equal to three, indicative of a mass fractal structure. Although more work is necessary to fully understand the subtleties of the PLLA-PEO-PLLA system, these differences might be related to the crystalline nature of the PLLA hydrophobic domains. WAXD studies (see Figure 3) on these gels show strong diffraction peaks at 2θ = 17 and 19 degrees corresponding to crystalline PLLA while the PRLA-based hydrogel showed no such peaks (32).



Figure 3. XRD showing crystalling PLLA in the hydrogel. (insets) Schematic representation of the self-assembled crystalline network along with a photograph of the oqaque hydrogel and a confocal microscopy image.

Conclusions PLA-PEO-PLA triblock copolymers have been extensively studied over the years, and have found use in a wide variety of applications. Understanding this copolymer system at a more fundamental level has provided insight into the connection between the molecular structure, self-assembled network structure, and bulk properties. Further study is required before a full understanding is availabe and the ability to predict the material properties can be made. A number of ways to influence the mechanical properties of the resulting hydrogel have been demonstrated including: at the monomer level by alternating the stereo-center, and at the nanometer-scale by covalently capturing the self-assembled structure.

Acknowledgments Data appearing in this review was supported by the central facilities of the NSF-funded Center for Hierarchical Manufacturing (CMMI-0531171) and the NSF-funded MRSEC on Polymers (DMR-0213695). We thank a number of agencies and companies for their support of the years including ARO, ONR, NSF, 3M, and DuPont.


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PLA-PEO-PLA Hydrogels: Chemical Structure, Self-Assembly and

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