Hydrophilic and Amphiphilic Polyethylene Glycol-Based Hydrogels

Nov 7, 2012 - 1H Magic Angle Spinning (MAS) NMR. 1H MAS ..... B and C), which exhibit different types of behavior are approximately delineated in Figu...
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Hydrophilic and Amphiphilic Polyethylene Glycol-Based Hydrogels with Tunable Degradability Prepared by “Click” Chemistry Vinh Truong,† Idriss Blakey,†,‡ and Andrew K. Whittaker*,†,‡ †

Australian Institute for Bioengineering and Nanotechnology, and ‡Centre for Advanced Imaging, The University of Queensland, Brisbane, 4072, Australia ABSTRACT: Hydrogels with tunable degradability have potential uses in a range of applications including drug delivery and tissue scaffolds. A series of poly(ethylene glycol) (PEG) hydrogels and amphiphilic PEGpoly(trimethylene carbonate ) (PTMC) hydrogels were prepared using copper-catalyzed Huisgen’s 1,3-dipolar cycloaddition, or “click” chemistry as the coupling chemistry. The fidelity of the coupling chemistry was confirmed using Fourier transform infrared (FTIR) and 1 H magic angle spinning (MAS) NMR spectroscopy while thorough swelling and degradation studies of the hydrogels were performed to relate network structure to the physical properties. The cross-linking efficiency calculated using the Flory−Rehner equation varied from 0.90 to 0.99, which indicates that the networks are close to “ideal” at a molecular level. However, at the microscopic level cryogenic scanning electron microscopy (cryo-SEM) indicated that some degree of phase separation was occurring during cross-linking. At 37 °C and pH 7.4, the degradation rate of the hydrogels increased with decreasing cross-link density in the network. Introduction of PTMC as the cross-linker produced an amphiphilic gel with higher cross-link density and a longer degradation time. The degradability of the resultant hydrogels could thus be tuned through control of molecular weight and structure of the precursors.

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INTRODUCTION The preparation of advanced medical devices, drug delivery systems, and sensing devices has progressed rapidly owing in many cases to the continued development of polymeric hydrogels. Poly(ethylene glycol) (PEG) hydrogels are among the most widely studied and used polymeric materials for biomedical applications, due to their biocompatibility and low toxicity.1 These polymers have also been approved by the U.S. Food and Drug Administration for a variety of clinical uses,2 and thus many PEG-based gels have been widely studied, especially for drug delivery applications.2−6 PEG hydrogels have also been used to modify biomaterial surfaces to provide protein resistance and to enhance surface biocompatibility, due to low levels of nonspecific binding to a range of biological molecules such as proteins and polypeptides.7,8 PEG hydrogels can be formed by cross-linking of polymer chains, through either physical or chemical means. PEG hydrogels prepared by physical cross-linking are usually free of impurities arising from initiator fragments, and therefore are favorable for biomedical applications.5 However, the polymer chains in physically cross-linked PEG hydrogels are connected through weak, reversible interactions such as van der Waals forces, hydrogen bonding, ionic, or hydrophobic interactions. Therefore, the gels can often revert to their sol phase by application of relatively small mechanical forces, or by changes in temperature and solvent conditions.9 Chemically crosslinked networks prepared by a free radical process, for example, by UV irradiation of PEG precursors and copolymers, also produce gels with low levels of impurities. However, there is © 2012 American Chemical Society

generally a high occurrence of defects within the hydrogel network such as loops and entanglements.2 This is a consequence of the conventional radical cross-linking mechanism, where cross-linking monomers can either react to form cross-links (the ideal case), loops, or remain unreacted (both nonideal). For this reason, it is difficult to ascertain with great accuracy the relationship between the concentration of crosslinking agent and properties of the resulting hydrogels, such as mechanical properties, rates of solute transport, and network porosity. PEG hydrogels can also be prepared by reaction of functionalized polymeric precursors with cross-linkers that possess complementary reactivity.5 Such complementary systems include, radical-based thiolene,10−12 Michael addition (e.g., maleimide−amine),13−15 1,3-dipolar cycloaddition of azides with ring strained alkynes,16,17 1,3-dipolar cycloaddition of azides with electron deficient alkynes18 and copper-catalyzed Huisgen’s 1,3-dipolar cycloaddition of azides with alkynes.19,20 The use of complementary functional groups for cross-linking eliminates the occurrence of loop-based defects, and the crosslink density can be controlled by changing the molecular weight of the polymeric precursors. The “click” chemistry reactions described above have been shown to be good candidates for the cross-linking reactions to prepare hydrogels, because of their high efficiency and selectivity. A consequence of more ideal Received: August 15, 2012 Revised: October 15, 2012 Published: November 7, 2012 4012

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OCH2CH2Cl, 4H), δ 3.62−3.65 (m, OCH2CH2O), 3.82−3.85 (t, OCH2CH2Cl, 4H). molar mass 2051, ĐM: 1.04. Synthesis of PEG-Bisazides (2). PEG-bisazides with Mn values of 1, 4, 6, and 20 kDa were prepared using the following procedure, where the use of the 2 kDa PEG-dichloride as a starting material is used as an illustrative example. Compound 1 (5 g, 25 mmol) and sodium azide (0.65 g, 0.1 mol) were dissolved in DMF (10 mL), and the mixture was stirred at 80 °C for 12 h. The solution was filtered and DMF was evaporated. The solid was dissolved in a minimum amount of DCM and precipitated into diethyl ether. This step was repeated twice, and the product was collected as a white powder (yield 91%). 1 H NMR (CDCl3): δ 3.59−3.61 (m, OCH2CH2N3, 8H), δ 3.62−3.65 (m, OCH2CH2O); molar mass: 2088; ĐM: 1.03. The yields of the reactions using the 1, 4, 6, and 20 kDa versions varied from 60% to 85% and the molar mass dispersity was in the range of 1.02−1.04. Synthesis of Tetrakis(hexynoate methoxide) (3). Pentaerythritol (1.36 g, 0.01 mol), hex-1-ynoic acid (5.6 g, 0.05 mol) and ptoluenesulfonic acid (0.192, 1 mmol) were mixed and heated to 160 °C, and the reaction was left stirring at this temperature for 12 h. The mixture was dissolved in DCM (50 mL), washed with saturated NaHCO3 solution (10 mL × 2) and brine (10 mL), and the organic phase was evaporated to dryness. The product was purified by column chromatography running hexane:ethyl acetate (4:6) on aluminum oxide (yield 58.6%). 1H NMR (CDCl3): δ 2.0 (t, CHCCH2, 4H), δ 2.48−2.62 (m, CHCCH2CH2CH2COO, 24H), δ 4.02 (s, CCH2OCO, 8H). Preparation of PEG Hydrogels Using “Click” Chemistry. A 1:1 molar ratio between the azide and alkyne groups was used for all hydrogels prepared. In a typical procedure, PEG-bisazide and tetrafunctional cross-linker were dissolved in a solution of ethanol/ isopropanol (1:1 volume ratio, 0.5 mL) to form a 50 wt/v% solution. To this solution was added sodium ascorbate (3 mg, 0.015 mmol) in water (0.25 mL), and the mixture was shaken vigorously until a clear solution was obtained. Copper sulfate (3 mg, 0.012 mmol) in water (0.25 mL) was added to the solution and was shaken for 10 s. The final polymer concentration was 25 wt/v%. This solution was added into a 5 mL vial, which had previously been silanized to prevent the gel adhering to the walls, and was left for 6 h at ambient temperature. The gel was then washed with ethylenediaminetetraacetic acid (EDTA) solution (0.2 M) and ethanol to remove copper ions and unreacted precursors. The dry gel was collected as a round tablet. The gel fraction was calculated as the weight percentage of dry gel over the total weight of the polymer precursors. Synthesis of 2, 2-Bis(hydroxyl methyl)propionate (4). To a 250 mL round-bottom flask was added 2, 2-bis(hydroxyl methyl)propionic acid (18 g 136.3 mmol), KOH (8.2 g, 146.6 mmol) and DMF (100 mL). The mixture was stirred at 100 °C for 2 h, then propargyl bromide (20.278 g, 137 mmol) was added dropwise over 30 min. The solution was stirred for 72 h, filtered, and the DMF was evaporated. The product was collected as a brown liquid (yield 81.9%). 1H NMR: δ 1.04 (s, CH 3CC, 3H), 2.43−2.45 (t, CHCCH2CO 1H), 3.62−3.66 (d, CH2OH, 2H), 3.78−3.82 (d, CH2OH, 2H), 4.65−4.66 (d, CHCCH2CO, 2H). Synthesis of 5-Methyl-5-propargylxycarbonyl-1,3-dioxane2-one (TMC-Alkyne) (5). Compound 4 (14 g, 0.081 mol) was mixed with ethyl chloroformate (17.65 g, 0.162 mol) and THF (100 mL) in a sealed vessel that was purged with argon and cooled in an ice bath. Triethylamine (16.44 g, 0.162 mol) in THF (20 mL) was added dropwise over 30 min under an argon atmosphere. The reaction was allowed to stir for 3 h, after which it was allowed to warm to 25 °C and left to stir overnight. The solution was then filtered, evaporated to dryness, and the product was precipitated in a mixture of ethyl acetate and diethyl ether (1:1) as white crystals (yield 57.3%). 1H NMR: δ 1.47 (s, CH3CC, 3H), 2.49−2.51 (t, CHCCH2CO 1H), 4.17−4.21(d, CH2OCO, 2H), 4.67−4.71 (d, CH2OCO, 2H), 4.75−4.76 (d, CHCCH2OCO, 2H). Synthesis of Poly(TMC-co-TMC-alkyne) (6). The ring-opening polymerization of TMC and TMC-alkyne was carried out in a nitrogen purged glovebox. In a typical reaction, TMC (0.765 g, 7.5 mmol) and

network structures is that the properties of the gels differ significantly from those prepared using noncomplementary coupling techniques, such as free radical cross-linking of acrylate-functionalized PEG chains. For example, a number of groups have demonstrated that PEG gels prepared through reaction complementary reactive groups have superior mechanical properties.19,21 One disadvantage of many strategies is that, in some cases, the procedures used for preparation of the polymer precursors required many consecutive timeconsuming synthetic steps. Introduction of hydrophobic polymer chains into hydrogel networks might offer several advantages for modifying the properties of hydrogels, such as increased solubilization of lipophilic molecules,22 as well as loading and delivery of hydrophobic molecules in conjunction with hydrophilic molecules,23 thus making the gels more versatile materials for drug delivery. Recently, trimethylene carbonate (TMC) has been used as a hydrophobic component in the preparation of amphiphilic PEG materials in order to control the degradability of the hydrogels.24,25 In these studies, PEG chains that were end-functionalized with TMC groups were cross-linked through copolymerization with TMC via organocatalyzed ring-opening polymerization to form amphiphilic PEG hydrogels with random network structures and distributions of the hydrophobic components. In this study, we have employed copper-catalyzed Huisgen’s 1,3-dipolar cycloaddition of azides with alkynes as the coupling reaction to form fully hydrophilic PEG hydrogels, as well as amphiphilic PEG-poly(TMC) (PTMC) hydrogels. To achieve this end, we have developed efficient synthetic pathways to prepare a number of the polymer precursors with clickable functional end-groups. The PEG-TMC networks differ from those reported by Nederberg and co-workers24,25 in that PTMC precursors with a small proportion of pendant alkyne groups were used as cross-linkers for PEG-α,ω-bis(azide)s. This series of polymers allows the properties of fully hydrophilic and amphiphilic gels to be compared. Taking advantage of the high efficiency of “click” chemistry and the complementary nature of the functional groups, the size and chemical composition of polymer chains within the hydrogel network can be precisely engineered, enabling the formation of hydrogels with tunable properties, such as swelling ratio and degradability. The swelling and degradation properties are related to the composition and molecular weight of the network components.

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EXPERIMENTAL SECTION

Materials. PEG with molecular weights of 2, 4, 6, and 20 kDa and molar-mass dispersity (ĐM)26 of 1.03, and all other chemicals except for TMC were purchased from Sigma Aldrich and used without further purification. TMC was purchased from Richman Chemicals and was recrystallized from ethyl acetate and dried under vacuum before use. Dichloromethane (DCM), tetrahydrofuran (THF), diethyl ether, and dimethylformamide (DMF) were made oxygen- and moisture-free using a purification unit under an inert nitrogen environment (MBraun Solvent Purification System Auto-5). Synthesis of PEG-Dichloride (1). PEG-dichlorides with Mn values of 1, 4, 6, and 20 kDa were prepared using the following procedure, where the use of the 2 kDa PEG as a starting material is used as an illustrative example. PEG having molar mass of 2 kDa (10 g, 50 mmol) was melted at 100 °C; thionyl chloride (0.9 g, 80 mmol) was then added dropwise over 10 min. The mixture was then stirred at 100 °C for 16 h and allowed to cool to room temperature. The solid was dissolved in a minimum amount of DCM and precipitated into diethyl ether. This step was repeated twice, and the product was collected as a white powder (yield 81.3%). 1H NMR (CDCl3): δ 3.37−3.39 (t, 4013

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Scheme 1. (A) Synthesis of PEG-bisazide; (B) Synthesis of Tetraalkyne Crosslinker; (C) Synthesis of PTMC with Pendant Alkyne Groups

TMC-alkyne (5) (0.495 g, 2.5 mmol) were mixed in a vial pretreated with Sigmacoat to eliminate surface-adsorbed water. 1,8-Diazabicycloundec-7-ene (DBU) (7.61 mg, 0.05 mmol) and benzyl alcohol (54.07 mg, 0.5 mmol) in DCM (5 mL) were added to the mixture. The reaction was stirred at room temperature for 12 h at which point TMC-alkyne was found to reach 99% conversion (by 1H NMR). The reaction was terminated by addition of 1 mL of benzoic acid in DCM (1 mg/mL). The solution was then added dropwise into cold methanol, and the product was collected as a white solid, which became transparent after drying (yield 78%). The composition of the TMC-alkyne in the copolymer of TMC and TMC-alkyne was 27.5 mol % as determined by 1H NMR. Molar mass 11850, ĐM: 1.47. 1H NMR (CDCl3): δ 1.23 (s, CH3CC), 2.26 (t, CHCCH2CO), 3.75 (m, OCH2CH2O), 3.92(d, CH2OCO), 4.56 (m, CH2OCO). Preparation of PEG-PTMC Hydrogel Using “Click” Chemistry. A 1:1 molar ratio between the azide and alkyne groups was used for all hydrogels prepared. In a typical procedure, PEG-bisazide 6 k and poly(TMC-co-TMC alkyne) were dissolved in DCM (0.25 mL) in a 5 mL vial to form a 100 wt/v% solution. Sodium ascorbate (3 mg, 0.015 mmol) in DMF (0.5 mL) was added to the vial, and the solution was shaken vigorously until a clear solution was obtained. Copper sulfate (3 mg, 0.012 mmol) in water (0.25 mL) was added to the solution, which was shaken for 10 s. The solution was poured into a 5 mL vial, which had been silanized beforehand to prevent the gel sticking to the walls of the vial, and was left for 6 h at 25 °C. The gel was then washed with EDTA solution (0.2 M), DCM, and ethanol to remove copper ions and unreacted residue. The dry gel was collected as a round tablet. (gel fraction = 0.95). Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) Measurements. ATR-FTIR spectra were obtained using a Nicolet Nexus 5700 FTIR spectrometer equipped with a Nicolet Smart Orbit accessory fitted with a single bounce, diamond internal reflection element (IRE) (Thermo Electron Corp., Waltham, MA). Spectra were recorded at 4 cm−1 resolution for at least 32 scans with an optical path difference velocity of 0.6289 cm s−1 and a scan range of 4000−525 cm−1. Solids were pressed directly onto the diamond IRE of the ATR accessory without further sample preparation. Spectra were manipulated using the OMNIC 7 software package (Thermo Electron Corp., Waltham, MA). 1 H and 13C NMR. 1H and 13C NMR spectra were collected using a 5 mm BBOz gradient probe at a temperature of 298 K on a Bruker Avance 300 MHz spectrometer. Deuterated solvents for NMR spectroscopy were commercially obtained (Cambridge Isotopes) and

were at least 99.8 atom % D. Chemical shifts (δ) are reported in parts per million (ppm) relative to residual solvents [δ(1H NMR) = 7.26 ppm and δ(13C NMR) = 77 ppm]. 1 H Magic Angle Spinning (MAS) NMR. 1H MAS NMR spectra were run on a Bruker AVANCE 700 MHz spectrometer operating at 700.1 MHz for 1H NMR. Gel samples, swollen in D2O, were packed into 4 mm partially stabilized zirconium oxide rotors, and spun in a 4 mm MAS probe at spinning speeds ranging from 2 to 3 kHz. The spectral width was 30 kHz, the excitation pulse length was 10 μs, and the recycle delay was 3 s. Swelling and Degradation Studies. The hydrogels were washed with an EDTA solution (0.01 M) and ethanol to remove unreacted PEG and copper residues, and were dried at 70 °C under reduced pressure until they reached constant weight prior to swelling measurements. For swelling measurements, the gel was immersed in distilled water at 37 °C. At certain time intervals, the hydrogel was removed, wiped with soft tissue to remove excess surface water, and the masses were measured. For the degradation studies, the dried polymers were transferred to a phosphate buffered saline (PBS) solution of pH 7.4 at 37 °C, and the mass measured periodically as described above. Cryogenic Scanning Electron Microscopy (Cryo-SEM). The swollen hydrogels were surface dried with a lint free tissue and fixed on carbon-filled TissueTek between two miniature rivets on a vacuum transfer rod. The samples were fractured under liquid nitrogen and transferred to a cryostat chamber, which was maintained at −170 °C. The top rivet was fractured to reveal the cross-sectional surface, which was coated with gold for 90 s at 1 mA. The sample was then transferred to the microscope chamber, which was also maintained at −170 °C. Hydrogel specimens were examined using a Phillips XL30 SEM with a 15 kV accelerating voltage operating under high vacuum. Size Exclusion Chromatography. The chromatographic system consisted of a 1515 Isocratic pump (Waters), a 717 autosampler (Waters), Styragel HT 6E and Styragel HT 3 columns (Waters) run in series, a light scattering detector DAWN 8+ (Wyatt Technology Corp.) and a 2414 differential refractive index detector (Waters). THF was used as the mobile phase at a flow rate of 1 mL/min. ASTRA (Wyatt Technology Corp.) and Empower 2 (Waters) were used for data collection and processing. For the determination of molar mass by conventional SEC, the columns were calibrated using polystyrene standards (Waters) covering the molar mass range of 3.07−1,320 kDa. A dn/dc of 0.130 was used for the analysis. This value was obtained for the polymers by running manually six samples of varying known 4014

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with EDTA was determined to be less than 1.5 μmol·g−1.30 Similar gels were also prepared using the 1, 4, 6, and 20 kDa PEG precursors. Synthesis of Amphiphilic PEG-PTMC Hydrogels. The amphiphilic hydrogels were prepared by the cross-linking reaction of the PEG bisazides with a multivalent hydrophobic cross-linker based on PTMC with alkyne functionalities pendant to the backbone (Figure 1 c). The polymeric crosslinker was prepared using a method adapted from Pratt et al.31 In this method, TMC was copolymerized with TMC-alkyne (5) to produce linear PTMC having pendant alkyne groups (6) (Scheme 1C), which are available for subsequent “click” chemistry reactions. In their previous study, Pratt et al.31 demonstrated that the alkyne-substituted TMC will be incorporated at a faster rate than TMC, and hence a degree of tapering of the copolymer is expected. However, without a detailed study of the relative reactivity of the monomers during the copolymerization, we are unable to predict the degree of tapering. The PTMC-alkyne copolymer of number-average molecular weight approximately equal to 12 kDa and with approximately 24 pendant alkyne groups was used as a hydrophobic cross-linker. Preparation of hydrogels containing hydrophobic PTMC was more difficult than preparation of PEG gels using the tetrafunctional cross-linker, due to the insolubility of the PTMC precursor in water. Copper(I) bromide in dimethyl sulfoxide (DMSO) was found to be an effective system for formation of amphiphilic PEG-PTMC gels, due to DMSO being a good solvent for both PEG and PTMC. The gel was formed in less than 30 min. However, the DMSO was difficult to remove from the gel following reaction. The presence of DMSO after cross-linking was confirmed using FTIR spectroscopy of the material even after extensive washing and drying at high temperatures (data not shown). In addition, DMSO forms a stable complex with copper(I) making copper removal more difficult. The gels prepared using Cu(I)Br in DMSO were seen to have a slightly yellowish color even after several washings with EDTA solution, as opposed to the colorless gels prepared from sodium ascorbate/CuSO4 in water/ethanol, indicating the presence of a significant amount of copper within the TMC-based gels. As an alternative, a solvent mixture of DCM, water, and DMF was chosen, because DCM enhances the solubilization of PTMC and the gel was easily purified by washing with EDTA solution. PEG-PTMC gels were readily formed using this solvent system and sodium ascorbate/CuSO4 as the catalyst system. The gels were colorless after washing with EDTA solution. Spectroscopic Characterization of Networks. ATRFTIR spectra of the starting materials and products of the hydrophilic PEG gels are shown in Figure 2. The tetraalkyne cross-linker exhibits characteristic bands at 3270 and 1725 cm−1 for the C−H stretch and CO stretching mode of the ester, respectively. The PEG bisazide precursor exhibits a characteristic peak at 2050 cm−1 for the out-of-phase NNN stretching mode of the azide end groups, as well as a spectroscopic fingerprint from 700 to 1500 cm−1 that is characteristic of PEG. The spectrum of the dried gel also has this spectroscopic fingerprint of PEG, which is expected, because the PEG forms the major component of the gel. The appearance of the CO stretch at 1725 cm−1 is indicative of the cross-linker being incorporated into the gel structure. The absence of the alkyne band at 3270 cm−1 and the azide band at 2050 cm−1 are indicative of the Huisgen’s 1,3-dipolar cycloaddition reaction being successful.

concentrations through the RI detector and measuring the detector response versus concentration. This value agreed with previously published dn/dc values in the literature.27,28

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RESULTS AND DISCUSSION Hydrogel Preparation. Synthesis of PEG Hydrogels. α,ωBishydroxyPEG was reacted with thionyl chloride to yield the bischloride (1) and subsequently the bisazide precursor (2) by reaction with sodium azide, with an overall yield over 70% (Scheme 1A). Using this procedure, PEG-bisazides with different molar masses were synthesized as precursors for the preparation of hydrogels. The cross-linker (3) was prepared by a one-step synthesis by condensation of pentaerythritol and hexynoic acid using toluene sulfonic acid as the catalyst at a reaction temperature of 160 °C (Scheme 1B). This process was also found to couple a range of organic acids including acetic acid, propionic acid and pentynoic acid to all four hydroxyl groups of pentaerythritol with high yield (from 70% to 75%), making it a versatile synthetic route for producing tetrafunctional building blocks from pentaerythritol. The PEG hydrogels were readily prepared from the PEG-bisazide and cross-linker in water/ethanol media with a yield of over 90% (Figure 1 b). Hydrogels with a gel content of over 90% were

Figure 1. Schematic diagrams showing (a) a key of the structures and functionality of precursors, and formation of the (b) PEG hydrogel and (c) amphiphilic PEG-PTMC hydrogel.

obtained at room temperature by reaction of PEG-bisazide and cross-linker in a molar ratio of 2:1, in the presence of 3 wt % sodium ascorbate and CuSO4. It should be noted that the amount of copper used may be considered high considering it was the catalyst; however, higher concentration of copper has previously been reported to give better yields in “click” chemistry.29 A gel was formed approximately 30 min after mixing and was allowed to stand in the mold for an additional 2−3 h before further purification and characterization. The fresh gel had a light blue color and became colorless after washing with an EDTA solution (0.2 M), which indicates that much of the copper was removed from the gel. In a similar study of PEG-containing gels, the copper content after washing 4015

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and the azide band at 2050 cm−1 demonstrates that Huisgen’s 1,3-dipolar cycloaddition reaction was also successful for this system. It should be considered, however, that the spectral sensitivity in the azide region is poor due to the strong absorbance of the diamond IRE, so 1H MAS NMR was used to gain a more accurate assessment of the reaction fidelity. 1 H MAS NMR spectroscopy of the fully swollen PEG hydrogels, in addition to solution-state NMR of the intermediates provides further insight into the efficiency of the “click” reaction during gel formation (Figure 3). A signal at 7.76 ppm in the 1H MAS NMR spectrum of the PEG6k hydrogel (Figure 4) can be attributed to the proton of the

Figure 2. ATR FTIR spectra of tetra-alkyne cross-linker (a), PEG6k bisazide polymer precursor (b), and the resultant gel after partial drying in the vacuum oven (c).

A similar set of observations were obtained for the hydrophobic PEG-PTMC gels, where the ATR-FTIR spectra for the starting materials and gel are shown in Figure 3. The

Figure 4. 1H MAS NMR of the fully swollen PEG6k hydrogel in D2O.

triazole ring formed by reaction of the azide and the alkyne groups.29 Peaks corresponding to the protons of the crosslinker (at 2.48−2.62 and 4.02 ppm) were also observed in the 1 H MAS NMR spectra of the hydrogel. A weak doublet signal assigned to the alkyne proton at 2.0 ppm is also present, indicating that the “click” reaction did not go to 100% completion, but this is expected for a network forming reaction, where functionalities can become physically isolated. Integration of the signal from the alkyne compared to the signal from triazole ring showed that the percentage of the unreacted groups is less than 4%. The gel content that was obtained at the completion of the reaction was over 90%, which demonstrated that the “click” reaction proceed to a high percentage of completion. Similar conclusions of high conversion of reactive groups can be deduced for the other hydrogels from the gel fraction measurements and calculated cross-linking efficiencies listed in Table 1. Microscopic Network Structure. The microscopic structure of the hydrogels during swelling was determined using cryo-SEM. The swollen PEG1k hydrogel appears to possess a continuous, relatively homogeneous structure at the magnifications used in our SEM (Figure 5a). For the remainder of the hydrogels, the cryo-SEM images show that at equilibrium swelling, the PEG and PEG-PTMC hydrogels have a porous and heterogeneous structure on the micrometer scale (Figures 5 and 6). For the PEG hydrogels, the pore size appears to increase in size as a function of the precursor molecular weight, but these pores are on the micrometer-scale, which is

Figure 3. FTIR spectra of PTMC-alkyne (a), PEG6k bisazide (b), and the resulting PEG-PTMC gel after partial drying in the vacuum oven (c).

PTMC with the pendant alkyne groups exhibits characteristic peaks at 3270 cm−1 for the C−H stretch of the alkyne and a CO stretch at 1740 cm−1 due to the carbonate group that forms part of the backbone. The spectral fingerprint observed between 800 and 1500 cm−1 is consistent with that of PTMC. The spectrum of the PEG-PTMC gel in this same region is a mixture of the PEG and PTMC spectral fingerprints. Furthermore, the absence of the alkyne band at 3270 cm−1 4016

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Table 1. Swelling Characteristics of PEG and PEG-PTMC Gels in Distilled Water (pH = 6.8) at 37 °C and Degradation Times in PBS at 37 °Cb gel

precursor M n (kDa)

yield (%)

swelling ratio

PEG1k PEG2k PEG4k PEG6k PEG20k PEG6k-PTMCa PEG20k-PTMCa

1 2 4 6 20 6 20

89 91 93 97 89 95 92

3.81 10.6 13.1 17.2 26 6.8 12.6

Mc kDa 0.472 0.987 1.97 2.95 9.02 n.d. n.d.

cross-linking efficiency C

diffusion coefficient (cm2sec−1)

degradation time (days)

0.94 0.98 0.99 0.98 0.90 n.d. n.d.

14.2 × 10−7 12.5 × 10−7 9.8 × 10−7 6.9 × 10−7

12 10 9 7 1 24 8

8.3 × 10−7 4.1 × 10−7

a PTMC with pendant alkyne groups were used as the cross-linker. bValues for Mc and crosslink efficiency for the PTMC gels were not determined, as described in the text.

Figure 5. SEM images of PEG hydrogels having different polymer chain lengths: (a) PEG1k, (b) PEG2k, (c) PEG4k, and (d) PEG6k after swelling in PBS for 24 h at 23 °C.

Casilda et al.39 have reported that the walls of macroporous networks consist of a nanoporous network in their cryogenic transmission electron microscopy (cryo-TEM) examination of networks of poly(N-vinylimidazole). Unfortunately, we were unable to perform higher resolution studies of our gels due to beam damage during analysis. The observation of a highly heterogeneous structure was initially unexpected, because the effective molecular weight between cross-links in these “click” gels is determined by the molecular weight of the narrow molar mass dispersity azidefunctionalized PEG chains. In particular, the spectroscopic analysis of the coupling reaction, and the measurement of residual non-network fraction, supported the formation of welldefined networks of accurately known values of Mc . However, as mentioned above, a number of other studies also report highly heterogeneous network structures.32−39 For example, recently Xu and co-workers38 have prepared a series of interpenetrating hydrogel networks via simultaneous “click” reaction of 4k diazide-PEG with tetrakis(2-

approximately 3 orders of magnitude larger than the mesh size of an ideal network of these polymers. Very similar morphologies have been observed during cryo-SEM analysis of hydrogels by a number of other groups,32−39 including for gels prepared using “click” reactions33,38 and controlled radical polymerization methods. 37 In particular, the group of Piérola34−36,39 has examined in detail the morphology of a number of hydrogels using SEM and related the observed structures to the progressive phase separation during network formation. For example, they have examined the effect of crosslinker content on the gel morphology in swollen networks of N,N-dimethylacrylamide cross-linked with N,N′-methylenebisacrylamide using conventional free radical initiators.34 The presence of a relatively high concentration of the cross-linker led to macrophase separation, and the size of the pores decreased with increasing cross-link density. This behavior was also observed in the current study (Figure 5) with a clear progression to smaller pores as the cross-link density increased (molecular weight between cross-links decreased). Calvino4017

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The swelling ratio, Q, was calculated from the following equation: Q=

Ws − Wd Wd

(1)

where Ws and Wd are the weights of the dry gel and fully swollen gel, respectively. The equilibrium polymer volume fraction, v2, which is the ratio of the dry gel volume and swollen gel volume, can be related to the swelling ratio Q as follows: ρs v2 = Qρp + ρs (2) where ρs is the density of swelling media, which is water, and ρp is the density of the dry gel. The number average molecular weight between cross-links, Mc was determined using the Flory−Rehner equation:40 v ̅ /V1[ln(1 − v2) + v2 + χ12 v2 2] 1 2 = − Mc Mn (v21/3 − (2/φ)v2)

(3)

Here, χ12 is the polymer−water interaction parameter which was reported to be 0.426 for PEG,41 V1 is the molar volume of water, v ̅ is the specific volume of the polymer, M n is the average molecular weight of linear chains before cross-linking, and φ is the functionality of the cross-linker. The cross-linking efficiency C and cross-link density νe can then be calculated using the following equations:42 ⎛M ⎞ C = ⎜ n ⎟ /fn ⎝ Mc ⎠

Figure 6. SEM images of PEG6k (a) and PEG6k-PTMC (b) after swelling in PBS for 24 h at 23 °C.

ve =

propynyloxymethyl)methane and ATRP of various hydrophilic monomers with PEG-diacrylate. The SEM image of the network formed from the cross-linking reaction of the diazide-PEG with the tetra-functional alkyne was also reported and, as in the current study, showed the presence of a macroporous network. The mechanism for formation of this macroporous network will be discussed in conjunction with the swelling studies that are detailed in the next section. When PTMC was incorporated in the PEG hydrogel network, the distribution of pore sizes was greatly broadened (Figure 6). Compared with the PEG gels that were made with PEG precursors of the same chain length, the PEG-PTMC networks have regions of significantly smaller pore size. This will effectively reduce the average pore size, and consequently the water sorption capacity, of the PEG-PTMC gels. To summarize the results on the characterization of the network morphology, it is clear that the network morphology can be modulated by either changing the PEG precursor size or by using a hydrophobic polymeric cross-linker. Introduction to Swelling Studies. The transport of solute molecules within polymer networks obviously occurs within the space filled by water. Therefore, the swelling properties of PEG hydrogels are particularly important for drug delivery applications. The kinetics of swelling and equilibrium properties will depend mostly on the chemical composition of the gel network and in some cases, the method of preparation, as well as environmental factors such as temperature, ionic strength and pH. In this section, we have examined the network properties of PEG gels with different chemical compositions based on their swelling profiles.

(4)

pg Mcf

(5)

where f n is the functionality of the polymer precursors and f is the functionality of the cross-linker. In this case, these values are 2 and 4, respectively. Effect of Chemical Structure on Swelling Behavior. The increase in mass of the PEG and PEG-PTMC hydrogels during swelling in water with a pH of 6.8 at 37 °C are shown in Figure 7. The results of the swelling studies of the PEG gels

Figure 7. Swelling ratio of PEG gels with different polymer chain lengths and PEG-PTMC gel in water at 37 °C. All samples had a similar thickness prior to swelling. (PEG1k: ▼; PEG2k: ■; PEG4k: ●; PEG6k: ▲; PEG6k-PTMC: Δ) The solid lines show the best fits of the data to the solution to Fick’s second law of diffusion into planar sheets. 4018

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phase separation determines to some extent the morphology of the gels at the micrometer scale. The influence of the hydrophobic segments becomes more important for the PEG-PTMC hydrogels. The TMC segments reduce further the aqueous solubility of the cross-linking moiety. The values of the molecular weight between cross-links for PEG-PTMC hydrogels could not be determined with reliability since we do not have an estimate of the interaction parameters of these gels with water. However, it is clear that the swelling ratios decreased when PTMC was incorporated into the gel structure. Therefore, introduction of the hydrophobic PTMC segments into the network reduces the capacity of the gel to accommodate water. Diffusion coefficients, D, were obtained by fitting the values of Wt/Wω against time to the solution of Fick’s second law of diffusion into a planar sample.45 The diffusion coefficient was found to decrease with decreasing cross-link density within the PEG gel network (Table 1). Similar relationships between cross-link density and diffusion coefficient of water have been reported in other studies, and are a consequence of the higher equilibrium swelling ratio of the gels with higher values of Mc .46,47 The higher swelling ratio leads to a slower approach of the system to fully swollen equilibrium, and hence a lower apparent diffusion coefficient. These results suggest that the diffusion coefficient of water and solutes can be controlled by varying the Mw of the polymer precursor. PEG-PTMC gels had a lower swelling ratio and higher diffusion coefficient compared to PEG gels with similar polymer chain lengths. This could be explained by the increase in cross-linking density in PEGPTMC due to the introduction of multiarm cross-linker PTMC-alkyne. Hydrolytic Degradation of the Networks. The hydrolytic degradation of the gels in PBS was monitored by following the water uptake over time at 37 °C. From the structure of the gels, it is expected that hydrolysis of the ester groups in the cross-linker will be the predominant mechanism for network degradation. The PEG and PEG-PTMC hydrogels exhibited different swelling behavior when the swelling media was changed from distilled water (pH 6.8) to PBS solution (pH 7.4), where three regions (A, B and C), which exhibit different types of behavior are approximately delineated in Figure 8. In region A, the gels were observed to swell quickly in PBS up to a quasi-equilibrium, which is similar to the behavior observed for swelling in distilled water. In region B, the gels were then observed to slowly imbibe water up to a second point.

with different polymer chain lengths and for the PEG-PTMC gels are summarized in Table 1. All samples had similar initial thicknesses, which was approximately 2.2 mm. The swelling ratio was observed to increase with increasing molar mass of the PEG bisazide precursors. This is mainly due to the decrease in cross-link density and corresponding increase in pore size within the network, which allows the gel to accommodate a larger number of water molecules. A swelling profile for PEG20k gel could not be obtained due to the difficulty in handling this sample. The PEG20k gel, which presumably has the lowest cross-link density, lost its structural integrity quickly after being immersed in PBS solution, making it difficult to accurately measure the weight. The molecular weight of the chains between cross-links, Mc , is an important parameter that determines how far a network can extend to accommodate solvent and solute molecules, and thus is a crucial property of the gel network for the potential application as a drug delivery system. This parameter was calculated using the Flory−Rehner equation as described above. The swelling ratio was found to increase with increasing molar mass of the PEG-bisazide. This is expected because longer PEG chains in the network should result in a lower cross-link density and larger pore sizes, which ultimately leads to greater water absorption. The value of the cross-linking efficiency, C, for PEG hydrogels (see Table 1) is within the range of 0.90 to 0.99, which indicates that networks formed were approaching an ideal structure. The most marked observation arising from the calculation of the molecular weight between cross-links, Mc , is that the values obtained for the PEG-only gels are consistently around 50% of the molecular weight of the fundamental building block, the bisazide-functionalized linear PEG chains. This result is contrary to expectations, because it corresponds to a larger than expected cross-link density. In many cases, in more conventional gels, the effective cross-link density is lower than expected from the stoichiometry of the reaction mixture, due to the presence of unreacted cross-linking moieties. Here we observe the opposite trend. The SEM results shed light on this observation, because they demonstrate a highly heterogeneous network structure possessing macropores, which become more pronounced when the cross-link density is lower. This suggests that phase separation is occurring during network formation. The PEG networks were prepared at 25 °C in a 1:1 mixture of alcohol (ethanol and isopropanol) with water. Alcohol was added to the reaction mixture to increase the solubility of the PEG chains and to solvate the pentaerythritol-based crosslinker, which is insoluble in water. At ambient temperatures water is a poor solvent for PEG chains compared to methanol, and the solvating power of water decreases with increasing molar mass.43 Of relevance also is the observation by Polverari and van de Ven44 that above a critical polymer concentration, aqueous PEG solutions consist of clusters and free polymer chains in thermodynamic equilibrium, and hence are inherently heterogeneous, albeit on a scale of tens of nanometres. As the polymerization proceeds, phase separation occurs and this is exacerbated by the presence of the strongly hydrophobic crosslinking groups. The molecular weight between cross-links decreases due to the reduced solubility of the polymer as conversion increases, and due to hydrophobic association of the cross-linking groups. Thus, despite the careful control of molecular weight between cross-links, the thermodynamics of

Figure 8. Swelling profiles of PEG hydrogels in PBS at 37 °C (PEG1k: ▼; PEG2k: ■; PEG4k: ●; PEG6k: ▲; PEG6k-PTMC: Δ) . 4019

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unreacted end groups remained. The cross-link density and swelling ratio of the hydrogels could be controlled by varying the molar mass and chemical composition of the polymer precursors. SEM studies indicate that the gels are structured by heterogeneous and interconnected pores, which are proposed to be formed by phase separation into polymer-rich and polymer-poor phases during cross-linking. Under physiological conditions, i.e., 37 °C and pH = 7.4, PEG hydrogels were found to degrade after reaching equilibrium swelling, where the degradation time was found to increase with increasing crosslink density (decreasing molecular weight between cross-links). Introduction of hydrophobic PTMC into the hydrogel networks produced amphiphilic hydrogels with a higher cross-link density and slower rate of degradation. The results show that by using this synthetic approach degradability can also be controlled by simply altering the molar mass and chemical composition of the starting materials. The ability to control the rate of network degradation and swelling ratio makes these materials potentially appealing in drug delivery applications. Studies of the release of several model drug molecules from these hydrogels will be reported in a forthcoming paper.

Following this there was a more rapid increase in the water uptake followed by a complete breakdown of the material. The second phase of water uptake when PEG hydrogels were swollen in PBS solution (Region B) can be attributed to the degradation of cross-link points by hydrolysis of the ester linkages within the network, which in turn increases the internal volume of the network and allows the gel to accommodate more water. Furthermore, there will be an increase in hydrophilicity going from an ester to a carboxylic acid and alcohol at the degradation sites. At higher pH, compared to the distilled water, the rate of degradation was enhanced due to the participation of base in the ester hydrolysis.48 If the slope of the increase in swelling in region B is used as a qualitative assessment of the breakdown of the network, then it can be seen that the breakdown of the network increases with increasing molecular weight of the precursor. This is a due to the number of cross-link points decreasing per unit volume for increasing molecular weight of the precursors. The lower number of cross-links leads to a higher water content and lower concentrations of degradable bonds within the network. Hence, it can be seen that the rate of network degradation can be tuned by selecting precursors of different molecular weights. The point of total dissolution also followed this trend (see Table 1), although the exact point of total dissolution was difficult to establish precisely, because the gels became fragile after extensive degradation had occurred. The PEG-PTMC hydrogels were found to only have slight increases in swelling, with no accelerated increase during the same time course of the degradation experiment, indicating that the degradation time was much longer than the PEG hydrogels. For example, the PEG6k-PTMC and PEG20k-PTMC was observed to degrade completely after 24 days and 8 days, respectively, while PEG6k and PEG20k hydrogels degraded after 7 days and 1 day, respectively, under the same conditions (see Table 1). The slower degradation rate of PEG-PTMC hydrogels compared to PEG hydrogels could be due to several reasons. When PTMC was used as the cross-linker, the crosslink density increased, and the swelling ratio of the gel decreased relative to the PEG gels formed with the same precursor molecular weight and using tetrafunctional crosslinker. Hence the carbonate linkages are less accessible to the hydrolyzing medium. In addition, the degradable bonds in PEG-PTMC networks are carbonate bonds, which have been shown to degrade less rapidly than the ester bonds49,50 in PEG networks. Finally the cross-link density of the PEG-PTMC networks is expected to be significantly higher than the PEGpentaerythritol networks, and therefore more bonds must be hydrolyzed to yield small molecule soluble components. Overall, these results demonstrate that by using a hydrophobic, highly functional PTMC-based cross-linker of high functionality, it is possible to increase the total degradation time of the gels.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +61-7-33463885; Fax: +61-7-33463973; E-mail address: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge funding for the International Biomaterials Research Alliance under the Queensland State Government National and International Research Alliance Program. Funding from the University of Queensland is also gratefully acknowledged. This work was also supported in part by the Australian Research Council (ARC) through ARC Linkage Infrastructure Grants LE0775684 and LE0668517, Discovery Project Grants DP0987407 and DP0878615, and Future Fellowship scheme FT100100721 (for I.B.).

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REFERENCES

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CONCLUSIONS In this study we have prepared a series of PEG-bisazides, a tetraalkyne cross-linker, as well as a novel alkyne-functionalized PTMC polymer cross-linker. Both polymer precursor and tetraalkyne cross-linker were synthesized in only one or two steps with high yields, making this an efficient way for preparing alkyne and azide materials used for preparing polymer networks. PEG-based hydrogels were subsequently prepared with high gel fractions and cross-linking efficiencies, and 1H MAS NMR was used to demonstrate that less than 4% of 4020

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