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Biomacromolecules 2009, 10, 128–133
Iron Tris(bipyridine) PEG Hydrogels with Covalent and Metal Coordinate Cross-Links Gina L. Fiore, Jessica L. Klinkenberg, Anne Pfister, and Cassandra L. Fraser* Department of Chemistry, University of Virginia, McCormick Road, P.O. Box 400319, Charlottesville, Virginia 22904-4319 Received September 6, 2008; Revised Manuscript Received November 9, 2008
Spontaneous gel formation of iron(II) tris(bipyridine)-centered poly(ethylene glycol) methacrylate ([Fe{bpy(PEGMA)2}3]2+) was observed without the addition of a cross-linking agent. BpyPEG2 macroligands were first modified with methacrylate groups using methacrylic anhydride and then combined with FeSO4 to produce [Fe{bpy(PEGMA)2}3]SO4. End group analysis by 1H NMR spectroscopy verified quantitative methacrylation of the PEG hydroxyl chain ends. A series of experiments and control reactions were performed to investigate the conditions required for gel formation. Hydrogels of [Fe{bpy(PEG-MA)2}3]SO4 were produced both in the presence and in the absence of a photoinitiator. Controls using MA-PEG-MA also formed hydrogels in the presence of [Fe(bpy)3]2+; however, the addition of a radical scavenger, TEMPO, prevented formation of a polymer network, suggesting radical involvement. Treatment of preformed hydrogels of bpy(PEG-MA)2 with aqueous solutions of FeSO4, CuBr2, and CoCl2 also produced materials with color changes indicative of complexation.
Introduction Poly(ethylene glycol) (PEG) is a water-soluble, U.S. Food and Drug Administration approved polymer used in biomedical applications due to its many beneficial properties.1-5 PEG can increase the solubility of biomolecules, decrease the immunogenic response, and prevent protein adsorption.2-10 PEG hydrogels are among the most commonly studied synthetic polymer networks and are used for applications in tissue engineering,5,6,9,11-16 drug delivery,6,11 and many devices, diagnostics, and sensors.6,17 Systems with physical, covalent, and metal coordinate cross-links are known. PEG-polyester (e.g., poly(lactide), poly(-caprolactone)) materials with physical cross-links9,18-22 are thermo-responsive gels20,23,24 that can be used as implants20 and for wound healing applications.23 Various methods exist for generating PEG gels with covalent cross-links. For instance, polymer chain ends can be modified with acrylate groups that can be polymerized by Michael-type reactions using sulfur cross-linking agents10,15,25-27 or via photoinitiation.15,28,29 PEG chains with methacrylate groups can also be photopolymerized and are preferred in certain applications due to the formation of stable radical intermediates.11,12,30 Degradable hydrogels have also been made from PEG-polyester (e.g., poly(lactide), poly(-caprolactone)) materials with acrylate and methacrylate chain ends. These materials, too, find application in cell encapsulation and tissue engineering.6,9,10,15,27,31,32 Metallohydrogels are also known. Sustained release of a model protein, hexa-histidine tagged green fluorescent protein, has been achieved from nickel iminodiacetic acid substituted hydrogels.33 Hydrogels with chelating ligands have been used for the removal of heavy metal ions (e.g., Cd2+, Hg2+, Pb2+) from aqueous media34 and Au3+ from HCl solution.35 Hydrogels with metal coordinate cross-links have also been reported.36-39 For instance, the combination of lanthanide metals and PEG oligomers with bis(2,6-bis(1′-methylbenzimidazolyl)-4-hydroxypyridine) pendant groups affords luminescent gels.37-39 Meth* To whom correspondence should be addressed. E-mail: fraser@ virginia.edu.
Figure 1. Schematic representation of hydrogel formation from [Fe{bpy(PEG-MA)2}3]2+.
acrylate-substituted polymeric metal complexes offer another route to hydrogels with both covalent and metal coordinate cross-links (Figure 1). This approach serves as the focus of this study and exploits the unique radical reactivity of [Fe(bpyPEG2)3]SO4 as the starting point for gel formation.40
Experimental Section Materials. Bipyridine-centered poly(ethylene glycol) (bpyPEG2) macroligands41,42 and [Fe(bpy)3]SO443 were prepared by previously
10.1021/bm800998g CCC: $40.75 2009 American Chemical Society Published on Web 12/09/2008
Iron Tris(bipyridine) PEG Hydrogels reported methods. Poly(ethylene glycol) (HO-PEG-OH, Mn ) 4.6 kDa) (Aldrich) was dissolved in toluene, dried by azeotropic distillation using a Dean-Stark trap, concentrated in vacuo, and then stored in a freezer in the drybox. Triethylamine (Aldrich) was dried over CaH2 and distilled under nitrogen prior to use. 2,2′-Bipyridine (Aldrich) was recrystallized (2×) from ethanol. Iron(II) sulfate (Strem) was titrated with 2,2′bipyridine (Aldrich) prior to use to verify the concentration of ferrous ions. Water (Fisher) was purged with argon prior to use. Chloroform-d (CDCl3) was passed through a short plug of dry, activated (Brockman I) basic alumina prior to use. Methacrylic anhydride (Aldrich), Irgacure 2959 (I-2959, Ciba), and all other reagents were used as received. Methods. 1H NMR (300 MHz) spectra were recorded on a Varian UnityInova 300 instrument in CDCl3, unless indicated otherwise. 1H NMR spectra were referenced to the signal for residual protio chloroform at 7.260 ppm. UV-vis spectra were taken in aqueous solution with a Hewlett-Packard 8452A diode-array spectrophotometer. Molecular weights were determined by gel permeation chromatography (GPC; THF, 25 °C, 1.0 mL/min) using multiangle laser light scattering (MALLS; λ ) 633 nm, 25 °C) and refractive index detection (λ ) 633 nm, 40 °C). Polymer Laboratories 5 µm “mixed C” columns along with Wyatt Technology Corp. (Optilab DSP interferometric refractometer, Dawn DSP Laser Photometer) and Agilent Technologies instrumentation (series 1100 HPLC) and Wyatt Technology software (ASTRA) were used in GPC analysis. Hydrogel formation was determined by the vial inversion method.44 PEG(C(O)C(CH3)CH2)2 (MA-PEG-MA).30 In a Schlenk flask, triethylamine (0.26 mL, 1.87 mmol) was added to a solution of HOPEG-OH (2.06 g, 0.450 mmol) in methylene chloride (10 mL). Methacrylic anhydride (0.27 mL, 1.82 mmol) was added dropwise to the PEG solution and the reaction mixture was stirred under nitrogen at room temperature in the dark for 4 d. The solution was then filtered through a neutral alumina plug, concentrated in vacuo, and precipitated (2×) into cold diethyl ether (-78 °C) to afford a white solid: 1.85 g (87%). 1H NMR (CDCl3, 300 MHz): δ 6.13 (s, RC(O)C(CH3)CH2), 5.57 (s, RC(O)C(CH3)CH2), 4.29 (m, RCH2CH2OC(O)C(CH3)CH2), 3.64 (m, PEG CH2CH2), 1.94 (s, RC(O)C(CH3)CH2). Methacrylation (NMR): 98%. Bpy{PEGC(O)C(CH3)CH2}2, 1. The macroligands were prepared as described above for MA-PEG-MA with the following reagent loadings: bpyPEG2 (1.00 g, 0.209 mmol, 4700 Da), triethylamine (58 µL, 0.42 mmol), methacrylic anhydride (123 µL, 0.834 mmol), and methylene chloride (10 mL). The product was precipitated from CH2Cl2/ Et2O (-78 °C), washed with additional cold Et2O, and dried in vacuo to afford a white solid: 0.941 g (94%). 1H NMR (CDCl3, 300 MHz): δ 8.64 (d, J ) 5.1 Hz, H-6, H-6′), 8.32 (s, H-3, H-3′), 7.37 (m, H-5, H-5′), 6.13 (s, RC(O)C(CH3)CH2), 5.57 (s, RC(O)C(CH3)CH2), 4.67 (s, bpyCH2), 4.30 (m, RCH2CH2OC(O)C(CH3)CH2), 3.64 (m, PEG CH2CH2), 1.95 (s, RC(O)C(CH3)CH2). [Fe{bpy(PEG-MA)2}3]SO4, 2. A solution of bpy(PEG-MA)2 (0.201 g, 0.035 mmol, Mn ) 5800) in H2O (5 mL) was added dropwise to an aqueous solution of FeSO4 · 7H2O (1.922 mL, 6.00 mM, 0.012 mmol), and the resulting mixture immediately turned pink. After stirring at room temperature under N2 for ∼15-20 min, the mixture was concentrated in vacuo. The crude product was precipitated from CH2Cl2/ cold Et2O (-78 °C), washed with cold Et2O, and dried in vacuo to provide the iron-centered polymeric complex as a pink solid: 0.177 g (88%). 1H NMR (CDCl3, 300 MHz): δ 8.64 (m, H-6, H-6′), 8.32 (m, H-3, H-3′), 7.37 (m, H-5, H-5′), 6.13 (s, RC(O)C(CH3)CH2), 5.57 (s, RC(O)C(CH3)CH2), 4.67 (s, bpyCH2), 4.30 (m, RCH2CH2OC(O)C(CH3)CH2), 3.64 (m, PEG CH2CH2), 1.95 (s, RC(O)C(CH3)CH2). UV-vis (H2O): λmax () ) 532 nm (8000 M-1 cm-1). Mn (calcd) ) 17500. [Fe{bpy(PEG-MA)2}3]SO4 Kinetics. A 1 cm quartz cuvette equipped with a Teflon-coated silcone septum was placed under Ar and charged with bpy(PEG-MA)2 (3 mg, 0.62 µmol, Mn ) 4700) in H2O (2.51 mL). A 3.14 mM aqueous solution of FeSO4 · 7H2O (65 µL, 0.21 µmol) was added and complex formation was monitored by UV-vis spectroscopy
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every 5 min for 2 h. The solution was then exposed to air for 1 min, resealed, and monitored every 30 min for 48 h. NMR Experiment. A 3 mL volumetric flask was charged with bpy(PEG-MA)2 (0.016 g, 0.003 mmol, Mn ) 4900) and FeSO4 · 7H2O (0.22 mL, 0.001 mmol, 4.86 mM) in D2O. The solution was stirred under air and monitored by 1H NMR spectroscopy. Bpy(PEG-MA)2 Hydrogel. Bpy(PEG-MA)2 (0.051 g, 0.010 mmol, Mn ) 4900) was dissolved in H2O (0.140 mL) in a vial. An aqueous solution of I-2959 (63 µL, 0.001 mmol) was added to bpy(PEG-MA)2 and irradiated with light (365 nm) for 30 min. [Fe{bpy(PEG-MA)2}3]SO4 Hydrogel. Method A. A vial was charged with [Fe{bpy(PEG-MA)2}3]SO4 (0.504 g, 0.029 mmol, Mn ) 17600), dissolved in H2O (202 µL), wrapped in foil, and left to stand overnight under ambient atmosphere. Method B. A vial was charged with bpy(PEG-MA)2 (0.051 g, 0.011 mmol, Mn ) 4800) and dissolved in H2O (15 µL). An aqueous solution of FeSO4 · 7H2O (194 µL, 0.004 mmol) was added and the vial was wrapped with foil and left to stand overnight under air. Method C. In a vial, bpy(PEG-MA)2 (0.051 g, 0.010 mmol, Mn ) 4900) was dissolved in H2O (0.139 mL). An aqueous solution of I-2959 (64 µL, 0.001 mmol) was added, and the mixture was irradiated with light (365 nm) for 30 min. After the hydrogel formed, an aqueous solution of FeSO4 · 7H2O (500 µL, 0.011 mmol) was added and the mixture was incubated overnight. Bpy(PEG-MA)2 Hydrogels. Other Metals. Hydrogels of bpy(PEGMA)2 were prepared as described above, substituting CuBr2 (500 µL, 0.010 mmol) or CoCl2 (500 µL, 0.017 mmol) for iron. MA-PEG-MA Hydrogel Controls. Photoinitiator. MA-PEG-MA (0.052 g, 0.012 mmol) was dissolved in H2O (0.50 mL). An aqueous solution of I-2959 (54 µL, 0.001 mmol) was added and the mixture was irradiated with light (365 nm) for 15 min. FeSO4. MA-PEG-MA (0.054 g, 0.012 mmol), H2O (0.40 mL), and an aqueous solution of FeSO4 · 7H2O (105 µL, 0.004 mmol) were combined in a vial. The mixture was allowed to stand overnight under ambient light. [Fe(bpy)3]SO4. MA-PEG-MA (0.054 g, 0.012 mmol), H2O (0.15 mL), and an aqueous solution of [Fe(bpy)3]SO4 (356 µL, 0.004 mmol) were combined, and the solution was allowed to stand overnight under ambient light and in the dark. Radical Inhibition. MA-PEG-MA (0.058 g, 0.013 mmol) was dissolved in H2O (0.25 mL). An aqueous solution of [Fe(bpy)3]SO4 (250 µL, 0.004 mmol) and the radical scavenger 1-piperidinyloxy (TEMPO) (0.003 g, 0.019 mmol) were added and the solution was left to stand overnight under ambient light.
Results and Discussion PEG materials, HO-PEG-OH, and bpyPEG2 macroligands were modified with methacrylate terminal groups, for use as precursors to polymer networks. We first attempted to prepare methacrylate modified materials by combining bpyPEG2 with methacryloyl chloride in the presence of Et3N, following reported methods.12,28 By this approach, 50-60% methacrylation was obtained, which is lower than literature values.12 Additionally, GPC analysis of reaction products before and after purification showed a broadening of the molecular weight distribution. The presence of high molecular weight species is indicative of spontaneous cross-linking of macroligand chain ends. To increase methacrylation and obtain stable bpy-centered dimethacrylate PEG macroligands (bpy(PEG-MA)2) (1), an alternative approach was explored. Work by Lin-Gibson30 showed that there are several benefits to using methacrylic anhydride instead of the acid chloride. The PEG chain ends were quantitatively functionalized to produce dimethacrylate PEG (MA-PEG-MA). Although reaction times for this procedure are longer (4 d vs 36 h), the use of a toxic lachrymator reagent, methacryloyl chloride, is avoided and fewer precipitation steps are required for polymer purification. Using a similar method, bpyPEG2 was combined with methacrylic
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Figure 2. 1H NMR spectrum of bpy(PEG-MA)2 in CDCl3.
anhydride and Et3N in CH2Cl2 solution, and the reaction was stirred at room temperature for 4 d in the dark (eq 1). Initial attempts employed 1.4 equiv of Et3N and 2 equiv of methacrylic anhydride; however, only 50-70% methacrylation was observed. When concentrations of methacrylic anhydride and Et3N were increased (4 equiv each), methacrylation of the PEG chain ends was nearly quantitative, as determined by 1H NMR analysis. Given the basic pyridyl sites in bpy macroligands, fewer equivalents of base (2 equiv Et3N) can be used in this reaction. The bpy(PEG-MA)2 products were purified by passage through a neutral alumina plug followed by precipitation from solution into cold diethyl ether to afford a white polymer product in good yield (∼85-95%).
Purified products were analyzed by 1H NMR spectroscopy, GPC, and spectrophotometric titration. The degree of chain end modification was determined via relative integrations of the vinylic hydrogens of the methacrylate end group (5.57 and 6.13 ppm) versus the protons alpha to the ester (-RCH2CH2COC(CH3)CH2 at 4.30 ppm) or the bpy methylene (bpyCH2 at 4.67 ppm) in 1H NMR spectra (Figure 2).12,30 In both cases, the relative integrations are 1:2, indicative of quantitative chain end modification. In GPC analysis of bpyPEG2 and HO-PEG-OH starting materials as well as their acrylated products, symmetric peaks, and low molecular weight distributions were obtained (PDIs < 1.05). Methacrylate PEG materials are known to cross-link in the presence of visible light under ambient conditions. This has been observed for bpy(PEGMA)2 too, as evidenced by a GPC trace with a broad distribution
Fiore et al.
Figure 3. Spectrophotometric titration for bpy(PEG-MA)2 (Mn ) 5800) and FeSO4 in aqueous solution to form [Fe{bpy(PEG-MA)2}3]SO4. Equivalents required to reach end point ) 3.0.
(PDI ) 1.75). Spontaneous cross-linking was averted by storing samples under an inert atmosphere in the drybox freezer (-29 °C). Uniform, narrow elution peaks and low PDIs (
