Investigation of Apatite Mineralization on Antioxidant

Aug 28, 2012 - The polymer–apatite composites were examined by electron scanning ... blocks for advanced polymers: synthesis, properties and applica...
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Investigation of Apatite Mineralization on Antioxidant Polyphosphazenes for Bone Tissue Engineering Nicole L. Morozowich, Jessica L. Nichol, and Harry R. Allcock* The Pennsylvania State University, Chemistry Research Building, University Park, Pennsylvania 16802, United States S Supporting Information *

ABSTRACT: Synthetic bone grafts that promote the natural mineralization process would be excellent candidates for the repair or replacement of bone defects. In this study, a series of antioxidant-containing polyphosphazenes were evaluated for their ability to mineralize apatite during exposure to a solution of simulated body fluid (SBF). All polymers contained ferulic acid (antioxidant), cosubstituted with different amino acid esters linked to the polyphosphazene backbone. Differences in the side groups determined the hydrophobicity or hydrophilicity of the resulting polymers. All of the polymers mineralized monocalcium phosphate monohydrate, a type of biological apatite. However, the mineralization process (the amount of deposition and length of time) was dependent on the hydrophilicity or hydrophobicity of the polymers. The polymer−apatite composites were examined by electron scanning microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, differential scanning calorimetry, and thermogravametric analysis. Weight gain data were also obtained. To verify that the nucleation process was due to the presence of calcium and phosphate, two standard solutions were prepared: one solution (NaCl solution) contained only sodium chloride, and the second solution (mSBF) was similar to SBF except without known crystal growth inhibitors such as Mg2+ and HCO3−. No mineralization occurred when the polymers were exposed to the NaCl solution, but mineralization took place upon exposure to mSBF. The apatite phase produced was hydroxyapatite (HAp). The mineralization process in mSBF was much more extensive, with all samples gaining more weight following exposure to SBF. A similar trend was also found (as in the case of SBF), with the amount of deposition and length of deposition time depending on the hydrophilicity/hydrophobicity of the polymer. These results suggest that the nucleation process is due to calcium and phosphate, and the absence of crystal growth inhibitors allows for the rapid nucleation of HAp. In both cases, the mineralization process was favored on hydrophilic surfaces (static water contact angle of 56−65°) versus hydrophobic surfaces (71−86°). KEYWORDS: antioxidant, polyphosphazene, biodegradable, bone tissue engineering, apatite, matrix hydrophobicity/hydrophilicity

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INTRODUCTION The development of synthetic scaffold materials that possess both the mechanical and chemical properties of bone would be a significant advance in the field of bone tissue engineering. Bone is composed of crystalline hydroxyapatite [Ca10(PO4)6(OH)2, HAp] mineralized onto highly aligned collagen fibrils, together with proteins commonly found in the extracellular matrix such as osteopontin and bone sialoprotein (BSP). The mechanism responsible for the formation of this complex architecture is unclear.1 However, it is thought to be due to the mechanical signals provided by the self-assembled collagen2 and/or the presence of the charged proteins in the extracellular matrix, which facilitate the nucleation of HAp.1,3,4 With respect to the charged proteins, the majority are decorated with acidic functional groups such as carboxylic acid units from aspartic and glutamic acid residues.5 These residues are believed to facilitate the binding of calcium and thus serve as nucleation points for HAp mineralization.1,3,4 Therefore, several researchers have focused on the design of synthetic materials decorated with charged functional groups to mimic the proteins present in the extracellular matrix.6−8 © 2012 American Chemical Society

Another factor rarely considered that may affect the mineralization process is the matrix hydrophobicity. For example, BSP and osteopontin are both rich in acidic residues; however, BSP is more efficient at promoting mineralization than osteopontin.3 This suggests there are other factors involved in the mineralization process, such as the matrix hydrophobicity and/or conformation. In order to study the mineralization behavior, polymers are exposed to a solution of simulated body fluid (SBF) with ion concentrations similar to those of human blood plasma. This has been shown to be an effective method for predicting the in vivo bone bioactivity.9 Specifically, polymer scaffolds that mineralized apatite had improved osteoconductivity and osteoinductivity compared to unmineralized polymers.10 Several polymers have been investigated for this application such as poly(ethylene glycol),7 bacterial cellulose,8 poly(L-lactic acid),11,12 and polycaprolactone.6 In each case, some type of Received: July 19, 2012 Revised: August 13, 2012 Published: August 28, 2012 3500

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Scheme 1. Polymer Synthesis of Antioxidant Polyphosphazenes (Ax-Ps)

ferulic acid (TCI/Sigma Aldrich), glycine ethyl ester hydrochloride (Chem Impex), alanine ethyl ester hydrochloride (Chem Impex), valine ethyl ester hydrochloride (Chem Impex), phenylalanine ethyl ester hydrochloride (Chem Impex), sodium hydride (60% in mineral oil, Alfa Aesar), tetrakis(triphenylphosphine)palladium(0) (Sigma Aldrich), thionyl chloride (Sigma Aldrich), morpholine (Alfa Aesar), sodium chloride (VWR), sodium bicarbonate (Aldrich), potassium chloride (JT Baker), dipotassium phosphate trihydrate (Acros Organics), magnesium chloride hexahydrate (JT Baker), HCl (EMD), calcium chloride (Aldrich), sodium sulfate (VWR), and tris(hydroxymethyl)aminomethane (THAM; Alfa Aesar) were used as received. Spectra/Por molecular porous cellulose dialysis membranes with molecular weight cutoffs of 12000−14000 were employed for purification of the polymers. Poly(dichlorophosphazene) was prepared by the thermal ring-opening polymerization of recrystallized and sublimed hexachlorocyclotriphosphazene (Fushimi Chemical Company, Japan) in evacuated Pyrex tubes at 250 °C.25 31P and 1H NMR spectra were obtained with a Bruker 360 WM instrument operated at 145 and 360 MHz, respectively. 31P NMR shifts are reported in ppm relative to 85% H3PO4 at 0 ppm. Glass transition temperatures were measured with a TA Instruments Q10 DSC apparatus with a heating rate of 10 °C/min and a sample size of ca. 10 mg. Decomposition temperatures (Td) were analyzed using a Perkin-Elmer PE-7 TGA unit with a heating rate of 20 °C/min under a constant flow of nitrogen atmosphere (50 mL/min) from 50 to 800 °C and a sample size of ca. 10 mg. Analysis was performed on Universal Analysis software. Gel permeation chromatography (GPC) was performed using a HewlettPackard 1047A refractive index detector and two Phenomenex Phenogel linear 10 columns. The samples were eluted at 1.0 mL/ min with a 10 mM solution of tetra-n-butylammonium nitrate in THF. The elution times were calibrated with polystyrene standards. Watercontact-angle (WCA) measurements were obtained using a RameHart contact-angle goniometer. Images were produced with “Snappy” video and analyzed using Image J. 1.2. Polymer Synthesis. The syntheses of ferulic allyl ester and polymers 2−5 were reported previously.21 Characterization data are provided in the Supporting Information. 1.2.1. Synthesis of Allyl-Protected Polymer 6. Poly(dichlorophosphazene) (1.00 g, 8.63 mmol) was dissolved in dry THF (100 mL). Ferulic allyl ester (2.22 g, 9.49 mmol) in dry THF (75 mL) was added to a suspension of sodium hydride (0.380 g, 9.49 mmol) in THF (75 mL). This mixture was allowed to react for 24 h at 40 °C and then added dropwise to the polymer solution. The mixture was stirred for 24 h at 40 °C and then refluxed for 24 h to obtain a partially substituted polymer. In a separate vessel, valine ethyl ester hydrochloride (6.27 g, 3.45 mmol) was suspended in THF (50 mL) with triethylamine (7.27 mL, 51.8 mmol). This suspension was refluxed for 24 h, filtered, and added to the polymer solution. The reaction mixture was stirred under reflux for 7 days. To this was added an additional 2.0 equiv of ferulic allyl ester (4.03 g, 17.3 mmol) and sodium hydride (0.690 g, 17.3 mmol) in 100 mL of THF. This mixture was refluxed for an additional 48 h and was subsequently filtered, concentrated, and precipitated into methanol (three times). 31P (145 MHz, CDCl3); δ −7.64, −22.5. 1H NMR (360 MHz, CDCl3); δ 7.36 (3H), 6.64 (1H), 6.13 (1H), 5.99 (1H), 5.22 (2H), 4.63 (2H), 3.87

biological apatite was formed, such as calcium-deficient HAp (cdHAp),8,11 dicalcium phosphate dihydrate (DCPD),13 carbonated apatite (cHAp),12,13 or HAp.7 However, because the biomimetic process occurs rather slowly (up to a few weeks), the mineralization experiments were conducted in higher than normal concentrations of SBF (5−10 times),11,13 the polymers were pretreated with high-concentration calcium phosphate solutions,6,7 or uneven mineral deposition was observed.8 One group of polymers that have only rarely been evaluated for in vitro bioactivity are polyphosphazenes. Polyphosphazenes are tunable inorganic−organic polymers with a backbone of alternating nitrogen and phosphorus atoms, with each phosphorus atom bearing two organic substituents.14 Specifically, amino acid ester containing polyphosphazenes15 have been utilized extensively for tissue engineering applications because of their good osteocompatibility,16−19 coupled with an absence of inflammatory response.20 More recently, a series of semihydrophobic to hydrophilic biodegradable antioxidantcontaining polyphosphazenes (Ax-Ps) have been synthesized (Scheme 1).21 Antioxidants are of interest because the administration of antioxidants at the site of a bone fracture can suppress the amount of free radicals produced and protect the surrounding tissue from further damage.22,23 These polymers are ideal scaffolding materials for bone tissue engineering because they are hydrolytically sensitive, and the antioxidant is released slowly during polymer hydrolysis. Also, the Ax-Ps contain approximately 50−80% of free carboxyl groups,21 which can bind calcium ions. Thus, the objective of the present study was to synthesize a range of hydrophilic to hydrophobic Ax-Ps and investigate the in vitro bioactivity by exposing the polymers to SBF. After SBF exposure, the polymer−apatite composites were characterized by electron scanning microscopy (ESEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). In addition, the method of nucleation was investigated by exposing the Ax-Ps to control solutions. The polymer− apatite composites (when applicable) were characterized by similar methods.

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MATERIALS AND METHODS

1.1. Reagents and Equipment for Polymer Synthesis. All synthesis reactions were carried out using standard Schlenk-line techniques and a dry argon atmosphere. The glassware was dried overnight in an oven at 125 °C before use. Tetrahydrofuran (THF) and triethylamine (EMD) were dried using solvent purification columns, with the final water content monitored by Karl Fisher titration.24 Dichloromethane (EMD), methanol (EMD), hexanes (EMD), ethyl acetate (EMD), allyl alcohol (Sigma Aldrich), trans3501

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1.5. Mineralization of Polymers in SBF. Polymers 2−7 were dissolved in DMSO (2.5 w/v %) and solution-cast into films (6 cm × 2.5 cm). The solvent was removed by lyophilization, followed by storage under vacuum for 1 week. The films were divided into eight samples (∼15 mg each) and were placed in 7.5 mL of SBF. The samples were maintained in a shaker bath at 37 °C for 4 weeks, with a daily exchange of the SBF solution. Two samples were removed every 1 week for each polymer, rinsed with deionized water several times, and dried by lyophilization. 1.5.1. Mineralization in mSBF and Control Experiments. Polymers 2−5 were prepared as described above. The films were divided into 12 samples (∼15 mg each). Four samples were placed in 7.5 mL of NaCl solution, mSBF, and deionized water. The samples were maintained in a shaker bath at 37 °C for 4 weeks, with a daily exchange of the respective solution. One sample was removed every 1 week for each polymer, rinsed with deionized water several times, and dried by lyophilization. 1.6. Equipment for the Characterization of Polymer− Apatite Composites. The samples obtained from SBF, a NaCl solution, and mSBF were analyzed each week by the methods described below. The morphology was determined by environmental ESEM. Images were obtained using a Phillips REI Quanta 200 electron scanning microscope. Low-vacuum mode was used for the imaging of uncoated samples under the following conditions: 20 keV of source voltage, pressure of 0.68 Torr, and a working distance of 12 mm. Energy-dispersive spectroscopy (EDS) was performed to determine the presence of calcium utilizing an Oxford EDS attachment and analyzed using INCA software. The Ca/P ratio was not calculated because of the polymer itself containing phosphorus. The mineral phase was determined by X-ray diffraction (XRD). XRD patterns were collected using a PANalytical X-Pert Pro MPD 2θ goniometer with Cu Kα radiation, fixed slit incidence (0.25° divergence, 0.50° antiscatter, and 10 mm specimen length), and diffracted (0.25° antiscatter, 0.02 mm nickel filter) optics. Samples were prepared by the flattening of the mineralized film onto a zero background holder using a glass slide. Data were collected at 45 kV and 40 mA from 5 to 50° 2θ using a PIXcel detector in scanning mode with a PSD length of 3.35° 2θ and 255 channels for a duration time of ∼20 min. The resulting patterns were analyzed with Jade+9 software and compared to patterns found in the International Center for Diffraction Data (ICDD) database and the Inorganic Crystal Structure Database (ICSD). The nucleation of various calcium phosphates was analyzed by FTIR using a Bruker Vertex V70 spectrometer (Bruker Optics, Billerica, MA) equipped with an MVP-Pro diamond single-reflection ATR accessory (Harrick Scientific, Pleasantville, NY). A total of 400 scans at 6 cm−1 resolution were averaged for each sample using a DTGS detector and a scan frequency of 5 kHz. In all cases, the spectrum of the clean diamond crystal was used as the reference spectrum. All spectral manipulations were performed using OPUS 5.5 (Bruker Optics, Billerica, MA). The thermal behavior of the polymer and mineralized composites was analyzed by DSC and TGA experiments described in section 1.1. The percent weight gain of the mineralized phase was determined by weighing samples before and after mineralization. Each sample was normalized using the weight loss obtained from the deionized water specimens.

(2H), 3.45 (3H), 1.62 (1H), 0.91 (3H), 0.58 (6H). Composition: 60% ferulic allyl ester and 40% valine ethyl ester. Mw: 1.3 × 106 g/mol (2955 repeat units). PDI: 3.23. Tg: 50 °C. Yield: 43.2%. 1.2.2. Synthesis of Allyl-Protected Polymer 7. Poly(dichlorophosphazene) (1.00 g, 8.63 mmol) was dissolved in dry THF (100 mL). Ferulic allyl ester (2.42 g, 10.3 mmol) in dry THF (75 mL) was added to a suspension of sodium hydride (0.414 g, 10.3 mmol) in THF (75 mL). This mixture was allowed to react for 24 h at 40 °C and then added dropwise to the polymer solution. The mixture was stirred for 24 h at 40 °C and then refluxed for 24 h to obtain a partially substituted polymer. In a separate vessel, valine ethyl ester hydrochloride (6.27 g, 3.45 mmol) was suspended in THF (50 mL) with triethylamine (7.27 mL, 51.8 mmol). This suspension was refluxed for 24 h, filtered, and added to the polymer solution. The reaction mixture was stirred under reflux for 8 days. To this was added by filter addition an additional 2.0 equiv of glycine ethyl ester hydrochloride (2.36 g, 17.3 mmol) and 3.0 equiv of triethylamine (3.61 mL, 25.9 mmol) after refluxing for 24 h. After completion, the mixture was subsequently filtered, concentrated, and precipitated into methanol (three times). 31P NMR (145 MHz, CDCl3): δ −0.36, −5.5, −17.5. 1H NMR (360 MHz, CDCl3); δ 7.50 (3H), 6.77 (2H), 6.22 (1H), 5.98 (1H), 5.27 (2H), 4.63 (2H), 4.0 (4H), 3.60 (5H), 1.62 (1H), 1.27 (6H), 1.00 (6H). Composition: 50% ferulic allyl ester, 25% glycine ethyl ester, and 25% valine ethyl ester. Mw: 4.8 × 105 g/mol (1204 repeat units). PDI: 2.20. Tg: 58 °C. Yield: 55.0%. 1.2.3. Deprotection of Polymers 6 and 7. The deprotection of polymers 2−5 was reported previously.21 Similar conditions were utilized for 6 and 7. Briefly, species 6 (0.500 g) was dissolved in 125 mL of THF. To this was added morpholine (0.967 g, 11.1 mmol) and tetrakis(triphenylphosphine)palladium(0) (0.128 g, 0.111 mmol). The solution was stirred for 22 h at room temperature and subsequently purified by dialysis versus 50:50 water and methanol (5 days) and by Soxhlet versus THF (4 days). 6. 31P NMR (145 MHz, DMSO-d6): δ −7.9, −23.2. 1H NMR (360 MHz, DMSO-d6): δ 7.40 (4H), 6.25 (1H), 4.05 (2H), 3.40 (3H), 1.23 (1H), 0.85 (9H). Tg: 125 °C. Td: 228 °C, 48.3% remaining at 800 °C. Yield: 98.0%. 7. 31P NMR (145 MHz, DMSO-d6): δ −1.2, −4.3, −18.0. 1H NMR (360 MHz, DMSOd6): δ 7.46 (3H), 6.35 (1H), 4.24 (2H), 3.93 (4H), 3.34 (5H), 1.45 (1H), 0.89 (12H). Tg: 102 °C. Td: 187 °C, 53.4% remaining at 800 °C. Yield: 98.0%. 1.3. Polymer Hydrolysis. The hydrolysis of polymers 2−5 was described in a previous publication and is provided in the Supporting Information.21 The procedure was repeated for polymers 6 and 7. Specifically, polymers 6 and 7 were dissolved in dimethyl sulfoxide (DMSO; 2.5 w/v %) and solution-cast into films (6 cm × 2.5 cm). Dried samples (∼10 mg) were placed in 5 mL of deionized water with a pH of 6.6. The samples were maintained at 37 °C in a shaker bath for 6 weeks. Each week, three samples were removed for each polymer. Weekly data are provided in the Supporting Information. 6: 17.5% loss ± 9.3. 7: 23% loss ± 6.5 (after 6 weeks). 1.4. Preparation of Control and SBF Solutions. 1.4.1. SBF Solution Preparation. A 1.5× SBF solution was prepared following the procedure reported by Kokubo et al.26 Briefly, analytical-grade NaCl, NaHCO3, KCl, K2HPO4·H2O, MgCl2·6H2O, 1 M HCl, CaCl2, and Na2SO4 were added sequentially to deionized water and buffered to a pH of 7.25 with THAM. The final concentrations of ionic species (in mM) were 213 Na+, 7.5 K+, 2.3 Mg2+, 3.8 Ca2+, 221.7 Cl−, 6.3 HCO3−, 1.5 HPO4−, and 0.8 SO42−. The solution was stored at 5 °C prior to usage. 1.4.2. Control Solutions (NaCl Solution and mSBF). Two control solutions were prepared with a pH of 7.25 using a THAM HClbuffered solution. The first (NaCl solution) was a solution of sodium chloride similar to the ionic concentrations reported in SBF (205 mM Na+ and 205 mM Cl−). The second was a modified SBF solution (mSBF) similar to SBF except free from Na+, Mg2+, HCO3−, and SO42−. The solution was prepared by the addition of K2HPO4 ·H2O, 1 M HCl, and CaCl2. The final concentrations of ionic species (in mM) was 7.5 K+, 3.8 Ca2+, 3.8 Cl−, and 1.5 HPO4−. The solutions were stored at 5 °C prior to usage.

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RESULTS AND DISCUSSION

Rationale for the Polymer Structure. The polyphosphazenes used in this work contained two different types of side groups. First, ferulic acid units (linked to the skeleton through aryloxy groups) provided antioxidant character, polymer stiffness, and pendent carboxylic acid moieties for the nucleation of calcium ions. Second, the ethyl esters of amino acids, glycine, alanine, valine, and phenylalanine were utilized to prevent water solubility of the polymers and for their ability to sensitize the polymers to hydrolysis. The rates of hydrolysis decreased with increasing hydrophobicity, in the order of Gly > 3502

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higher percentage of the aryloxy substituent showed higher molecular weights because this will limit the exposure to HCl generated from the amino acid ester side groups. Also, the shielding ability of the aryloxy substituent would further contribute to protection of the backbone. The glass transition temperatures (Tg) of polymers 2−7 (Table 1) ranged from 102 to 132 °C, the highest reported for a biodegradable polyphosphazene. It is believed that the carboxyl groups form a hydrogenbonded network, thus reducing the backbone freedom. In some cases, a melting temperature (Tm) was detected where the aryloxy substituents align, probably by π−π stacking. The decomposition temperatures (Td) of polymers 2−7 ranged from 187 to 255 °C. Polymers with a larger percentage of the aryloxy substituent generally had higher Td’s, probably because of the high stability of the aryloxy substituents. For example, polyphosphazenes that contain only aryloxy substituents have Td’s that range from 350 to 400 °C, while those with amino substituents have Td’s of 150−200 °C.28 Mineralization Experiments in SBF and Composite Characterization by ESEM. The polymers were first treated with 1.5× SBF for a period of 4 weeks. The time period of 4 weeks was chosen to allow adequate time for mineralization. A dynamic SBF treatment (solution changed every 24 h) was utilized because this has been shown to be the most effective method for mineralization.8 Swelling was observed during the treatment time with polymers 2−5 but not 6 or 7. Mineralization occurred on all polymers; however, the rate of deposition was different for each polymer. The polymers were found to mineralize in the following order from fastest to slowest, being 2 = 3 > 4 > 5 > 6 > 7. After soaking in SBF for 1 week, all of the polymers showed different levels of mineralization as revealed by ESEM, as shown in Figure 1. Polymers 2−4 had extensive mineralization; however, 5 and 6 had minimal mineralization, and 7 remained unchanged. The extensive mineralization revealed a uniform, porous inorganic coating. There were no definable surface features, and the composites were classified as amorphous. The inorganic coating was initially identified as a type of apatite because EDS measurements revealed the presence of calcium and phosphorus. However, the specific apatite phase was not determined by the Ca/P ratio because of the porous nature of the composites, which possibly exposed some uncoated polymer and its constituent phosphorus. Other techniques were utilized to identify the apatite phase, and these will be discussed later. Polymer 5 also yielded a porous inorganic coating like 2−4; however, it was heterogeneous. Polymer 6 had crystalline aggregate deposits with nonuniform geometries and platelike deposits of various sizes. These morphologies are consistent with monocalcium phosphate monohydrate (MCPM), a type of biological apatite.29 After 2 weeks in SBF, polymers 2 and 3 were no longer porous because a dense layer of apatite appeared to have mineralized through the pores observed earlier in week 1. The morphology was still mostly amorphous, but few crystalline deposits were evident. All other polymers remained unchanged. At 3 weeks in SBF, polymer 5 showed the same behavior as that of polymers 2 and 3, revealing a dense layer of apatite: however, it remained heterogeneously distributed on the surface. Polymers 2, 3, and 6 had more crystalline deposits, and polymer 7 began to show a few crystalline platelike deposits. All polymers at 4 weeks appeared to be the same as they were at week 3.

Ala > Val > Phe. Also, the hydrolysis rates can be controlled by varying the ratio of ferulic acid to amino acid ester. Polymer Synthesis. Polymers 2−5 were synthesized following a previously published procedure21, and 6 and 7 were synthesized with slight modifications. As described in our previous publication, traditional polyphosphazene synthesis typically occurs in two steps. First, the reactive intermediate, poly(dichlorophosphazene) (1), is generated by the ringopening polymerization of hexachlorocyclotriphosphazene. Second, the chlorine atoms are replaced by the reaction with the nucleophiles (ferulic allyl ester and an amino acid ester) in a two- or three-step sequential addition.21 For example, polymers 6 and 7 were synthesized by a three-step procedure. In each case, ferulic allyl ester was added first because of generation of the benign side product NaCl. Then the amino acid ester was added second to minimize the liberation of HCl, which can cause chain cleavage. Triethylamine was added as the hydrogen chloride acceptor to further minimize this effect. In the final step, either ferulic allyl ester (P6) or glycine ethyl ester (P7) was added to complete the substitution. Substitution was monitored by 31P NMR spectroscopy, and all reactions were complete within 4−13 days. Longer reaction times were required to ensure full substitution of the bulky nucleophiles. After nucleophilic substitution reactions were complete, the allyl-protecting groups were removed by treatment with morpholine and tetrakis(triphenylphosphine)palladium(0). This method has been utilized successfully to obtain polymers with free carboxyl groups.21,27 Polymer Structural Characterization. The polymers were characterized by 1H NMR spectroscopy, GPC, DSC, and TGA techniques. Structural characterization data can be found in the Supporting Information for polymers 2−5 and in the experimental section for polymers 6 and 7. Side-group ratios were determined by 1H NMR spectroscopy. Polymers 2−5 had 75−80% ferulic acid, polymer 6 60%, and polymer 7 50%. The difference in side-group chemistry provided polymers that were hydrophilic, semihydrophobic, and hydrophobic. This was estimated by WCA measurements, which can be found in Table 1. A lower percentage of ferulic acid (P6 and P7) Table 1. Static WCA Measurements for Ax-Ps and Thermal Characterization Information (DSC and TGA) P

WCA (deg)

Tg (°C)

Tm (°C)

2 3 4 5 6 7

56 63 71 71 78 82

116.4, 191.5 122.9 132 125 125 102

234.3 218.4 none none none none

Td, % remaining (at 800 °C) 235, 237, 241, 255, 228, 187,

56.0 54.3 48.9 50.6 48.3 53.4

resulted in more hydrophobic polymers. Also, polymers with hydrophilic/neutral amino acid esters [glycine (P2) and alanine (P3)] were more hydrophilic than those with hydrophobic amino acids [valine (P4) and phenylalanine (P5)]. This had a dramatic effect on the mineralization behavior. All allyl-protected polymers had relatively high molecular weights ranging from 4.8 × 105 to 3.2 × 106 g/mol. The molecular weights of the deprotected polymers could not be determined because of their insolubility in THF. However, they are expected to be similar because no degradation was detected during deprotection, with the lack of phosphate peaks at 0 ppm as analyzed by 31P NMR spectroscopy. Polymers containing a 3503

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Figure 1. ESEM images of polymers 2−7 after exposure to SBF for 3 weeks. Note: There was no change from week 3 to week 4 in all samples.

The difference in the mineralization behavior can be explained by variation of the side-group chemistry. There are two main differences with respect to the side groups: (1) polymers 2−5 contain 75−80% carboxylic acid groups, whereas polymers 6 and 7 contain only 50−60% and (2) polymers 2 and 3 contain hydrophilic/neutral amino acids, whereas polymers 4−7 contain hydrophobic amino acids. These differences create hydrophilic polymers (2 and 3), semihydrophobic polymers (4 and 5), and hydrophobic polymers (6 and 7). On the basis of the ESEM results, hydrophilic polymers (2 and 3) perform better than hydrophobic polymers (4−7) because a dense layer of MCPM forms within 2 weeks. This is reasonable because the initial nucleation process begins with the binding of calcium ions to carboxylic acid functional groups.1 These results were further confirmed by weight gain measurements. Weight Gain after Mineralization in SBF. Polymers 2−7 were tested for their weight gain after exposure to a solution of SBF for 4 weeks. Preweighed samples of the polymer were exposed to SBF, the solution was exchanged daily, and after each week, samples were removed, rinsed, and lyophilized. Before ESEM evaluation, the materials were weighed. All samples were normalized to the percent mass loss obtained from exposure of the polymers to a solution of deionized water, also exchanged daily. The results are shown in Chart 1. Polymers 2 and 3 gained the most weight, with 19 and 27%

Chart 1. Percent Weight Gain of MCPM of Polymers 2−5 upon Exposure to SBF for 4 weeks

weight gains, respectively. Polymers 4 and 5 gained 17 and 14%, respectively, and polymers 6 and 7 had less than 1% weight gain. Mineralization in Control Solutions and Composite Characterization by ESEM. After incubation in SBF, the polymers were exposed to standard solutions (NaCl solution 3504

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Figure 2. ESEM images of polymers 2−5 after exposure to mSBF for 2 weeks. Note: Only polymers 2−5 were tested, and all polymers exhibited heterogeneous deposition.

and mSBF) to confirm that the nucleation involved coordination of calcium and phosphate to the side groups on the polymer. However, polymers that showed little to no MCPM deposition (6 and 7) upon exposure to SBF were excluded from examination in the control solutions. At all time points, there was no mineral deposition during exposure to the NaCl solution. However, incubation in mSBF produced a nonuniform mineral coating on polymers 2−4 within 1 week and on polymer 5 within 2 weeks. The morphology of the deposited particles consisted of spherical globules of various sizes (Figure 2). This morphology is consistent with a bonelike apatite morphology resembling either HAp,7 cdHAp,11 or cHAp.13 The specific type of apatite phase was determined by XRD and FTIR. Weight Gain after Mineralization in mSBF. The weight gain of HAp was investigated after exposure of the polymers to mSBF for 2 weeks. The results were normalized to the percent mass loss obtained from exposure to deionized water. The mineralization process was faster and more extensive in mSBF than in SBF. More deposition of HAp on polymers 2−5 occurred from mSBF than MCPM deposition from SBF. After 2 weeks, a 13−43% weight gain of HAp occurred, as shown in Chart 2. More weight gain occurred with hydrophilic polymers (2 and 3) than with semihydrophobic polymers (4 and 5), as observed previously. Phase Identification of Mineralized Polymers by XRD. Following ESEM evaluation, polymers 2−7 were characterized by XRD to identify and confirm the type of nucleated apatite phase from SBF (MCPM) and mSBF (HAp) media. XRD patterns for mineralized polymers 2−5 and polymers 6 and 7 in SBF are shown in Figures 3 and 4, respectively. Before mineralization, all polymers contained a predominant broad peak at 2θ = 19−28°, centered at 23°, and a small broad peak at 2θ = 40°. After incubation in SBF for 1 week, all polymers except polymer 7 had a peak at 2θ = 7.5°, together with the 2θ

Chart 2. Percent Weight Gain of HAp of Polymers 2−5 upon Exposure to mSBF for 2 weeks

reflection at 23°. Reflections at both 7.5° (010) and 22.9° (120) are characteristic of MCPM [CaH2PO4·H2O, ICDD 0411-3010, major 2θ = 7.53 (010 hkl), 22.9 (120 hkl), and 24.1 (1−20 hkl)]. The reflection at 23° is broader than the reflection at 7.5° because it also contains the reflection attributed to polymers 2−5. There were no changes in the reflections after 2 weeks in SBF, and a representative example is shown for polymers 2−5 (Figure 3) and 6 (Figure 4) at 2 weeks. The reflections are broad because of the poor crystallinity of the deposited inorganic phase. This was supported by the lack of crystallinity in the ESEM images shown in Figure 1. After 3 weeks in SBF, the 2θ peak at 7.5° became much more intense for polymers 2−5, which could be due to the deposition of more MCPM or due to MCPM becoming more crystalline. Also, polymer 7 exhibited a peak at 2θ = 7.5°. These results 3505

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Figure 3. Representative XRD patterns of polymers 2−5 at 0, 2, and 4 weeks of mineralization in SBF. The specific polymer shown is polymer 2.

Figure 5. Representative XRD patterns of polymers 2−5 after 2 weeks of mineralization in mSBF. The specific polymer shown is polymer 3. Top: 2θ = 20−50°. Bottom: 2θ = 5−50°. Figure 4. XRD patterns of polymers 6 and 7 at 2 and 4 weeks of mineralization in SBF. The specific polymer shown at 0 weeks is polymer 6. Note: There was no mineralization observed for polymer 7 at 2 weeks.

examples in the literature, the 2θ peaks for nucleated and deposited HAp are broad.6,7,12,13 For example, there are three intense peaks for HAp at 2θ = 31.7° (211), 32.2° (112), and 32.9° (300), which appear as one broad peak centered at 31.7°, as shown in Figure 5. The overlap of the (211), (112), and (300) diffraction planes is indicative of poorly crystalline HAp.30 Analysis of Calcium Phosphate Nucleation by FTIR. FTIR evaluation of the polymers and polymer−apatite composites corroborated the nucleation of various calcium phosphates to the polymer. Shown in Figure 6 is a representative example (polymer 2) for polymers 2−6 after nucleation in SBF for 2 and 4 weeks. Note that polymer 7 showed no change in FTIR until week 3. Four main differences are notable before and after mineralization. (1) A decrease in the band at 1697 cm−1 and the appearance of two new bands at 1538 and 1385 cm−1 (Figure 6). This is due to the interaction of calcium ions with the carboxylic acid groups to form calcium carboxylate species. Calcium carboxylate species have two characteristic shifts at 1538 and 1385 cm−1.31 There was no doubt that the coordination occurred in the presence of calcium ions because no change could be detected by FTIR after nucleation in the standard NaCl solution at all weeks. (2) A band appeared at 3355 cm−1, which was attributed to the presence of the hydrate in MCPM (νOH mode; Figure 6).32 (3) The appearances of bands at 1236 cm−1 (δOH in-plane deformation) and 1072 cm−1

were confirmed by the ESEM images shown in Figure 1, with the appearance of crystalline deposits at week 3. There were no changes in the XRD patterns from 3 to 4 weeks, and a representative example is shown for polymers 2−5 (Figure 3) and 6 and 7 (Figure 4) at 4 weeks. Because of the slow mineralization of polymers 6 and 7, they were not examined in the control solutions. Results obtained in the mSBF solution for polymers 2−5 were different from those in the SBF solution. This was also seen by ESEM. Before mineralization, all polymers had a large broad peak at 2θ = 19− 28°, centered at 23°, and a small broad peak at 2θ = 40°. After incubation in mSBF for 1 week, all of the polymers except polymer 5 had peaks at 2θ = 25.8°, 31.7°, 39.8°, and 46.7°, together with the 2θ reflection at 23°, as shown in Figure 5. Reflections at 25.9° (002), 31.7° (211), 39.8° (130), and 46.7° (222) are characteristic of hydroxyapatite [HAp, Ca10(PO4)6(OH)2, FIZ 97849, major 2θ = 25.9° (002 hkl), 31.7° (211 hkl), 32.2° (112 hkl), 32.9° (300 hkl), 34.0° (002 hkl), 39.8° (130 hkl), 46.7° (222 hkl), 49.5° (123 hkl)]. After 2 weeks in mSBF, polymer 5 also had peaks characteristic of HAp. The increase in the incubation time for polymers 2−4 resulted in enhanced intensities in the 2θ peaks. Like many 3506

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FTIR results obtained from polymers 2−5 after nucleation in mSBF are presented in Figure 8. The results obtained by FTIR

Figure 8. Representative FTIR of polymers 2−5 after 2 weeks of mineralization in mSBF. The specific polymers shown are 2 (purple) and 3 (blue). Overlaid with HAp (as received) and the starting polymer (polymer 3).

further support the formation of HAp from mSBF. Polymers 2 and 3 are given as representative examples. Like the previous example after nucleation in SBF (Figures 6 and 7), the carboxylic acid stretch at 1697 cm−1 disappeared and two carboxyl bands appeared at 1539 and 1385 cm−1.31 After 2 weeks in mSBF, two major peaks appeared at 1015−1022 and 549−552 cm−1. These peaks correspond to the major bands for phosphate in HAp (ν3PO mode and ν4PO mode).34 The spectrum of HAp was overlaid with the mineralized polymers to more clearly show the major stretching vibrations of HAp (Figure 8). The major difference in the FTIR spectra between polymers 2 and 3−5 is that polymer 2 generates mainly HAp vibrations, whereas polymers 3−5 contain vibrations from the polymer and HAp. This can be explained by the nonuniform coating deposited on the polymers. Thermal Behavior of Composites Mineralized in SBF. The thermal properties of the mineralized polymers obtained from SBF were investigated briefly and are summarized in Table 2. These results further support the mineralization of

Figure 6. Representative FTIR of polymers 2−6 at 2 and 4 weeks of mineralization in SBF. The specific polymer shown is polymer 2. Top: 4000−500 cm−1. Bottom: 1850−400 cm1. Overlapped with the starting polymer.

(PO2 symmetrical stretch) are due to the presence of MCPM (Figure 7).32 (4) There are several shifts in the polymer bands

Table 2. Thermal Properties of Polymers 2−5 after 2 weeks of Mineralization in SBF and Polymers 6 and 7 after 4 weeks of Mineralization in SBFa polymer 2 3 4 5 6 7

Figure 7. Representative FTIR of polymers 2−7 after 4 weeks of mineralization in SBF. The specific polymer shown is polymer 3. Overlapped with MCPM and the starting polymer.

to that of MCPM. For example, the band at 914 cm−1 shifts to 922 cm−1, closer to that of MCPM at 948 cm−1 [P(OH)2 asymmetric stretch at 962 cm−1 and symmetric stretch at 914 cm−1]32 (Figure 7). Also, the vibration at 841 cm−1 shifts to that of MCPM at 850 cm−1 (γOH out-of-plane deformation).32 The shifts in the polymer peaks to those of MCPM can be explained by the ionic interaction of the nucleated apatite phase with the polymer. This behavior has been reported before with polymer−HAp composites, where OH groups in HAp interact with specific functional groups on the polymer and change the vibrations of the OH groups of HAp.33 Another explanation could be that the formation of a new composite changes the refractive index properties of the two individual components.

Td (°C), % remaining (at 800 °C) 302, 299, 297, 309, 262, 199,

60.6 58.3 53.8 55.0 49.6 54.0

increase in Td (°C)

Tm (°C)

67 62 56 54 34 12

71/245 80/238 80/258 90/257 89 87

a No T g transitions were observed for any polymers after mineralization because of the overlap with the large and broad endothermic transition.

hydrated calcium phosphate. There are three distinct differences after mineralization. (1) The thermal decomposition temperature (Td) increases because of nucleation and subsequent mineralization of calcium phosphate. This can be seen in Figure 9, where polymer 2 is shown as an example before and after mineralization. After exposure to SBF for 2 weeks, the Td of polymer 2 increases by 67 °C. This is the 3507

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was formed.39 When considering polymers 2−5, they contain 80% ferulic acid and 20% amino acid ester. The hydrolysis study of polymers 2−5 resulted in 10−25% weight loss after 6 weeks, while releasing ferulic acid.21 Hydrophilic polymers (2 and 3) hydrolyzed faster, 17−25%, than hydrophobic polymers (4 and 5), 10−13%. The release of ferulic acid may have promoted the formation of MCPM instead of other phases, similar to the behavior of citric acid. The constant release of ferulic acid may have also prevented the formation of HAp. Polymers 6 and 7 contain 50−60% ferulic acid and 40−50% amino acid ester. A pH-dependent hydrolysis study was carried out to determine the percent weight loss. Polymer 7 hydrolyzed at a faster rate (23%) than polymer 6 (17.5%). This result was expected because polymer 7 contains a small, hydrophilic, amino acid ester. As mentioned earlier, polymers containing smaller amino acid esters hydrolyze at a faster rate than polymers with larger amino acid esters.15 Both polymers also mineralize MCPM, but to a lesser extent than polymers 2−5. This is due to the difference in side-group chemistry, with polymers 6 and 7 containing only 50−60% of the carboxylic acid functional groups capable of binding calcium ions. It is suggested that the release of ferulic acid promotes the formation of MCPM in SBF but that does not explain why HAp precipitates from mSBF. The difference in the two solutions is that mSBF is nominally free of crystal growth inhibitors Mg2+ and HCO3−, which prevent the formation of HAp. It is believed that Mg2+ and CO32− enter the crystal structure of HAp and create a structural mismatch that causes the dissolution of HAp.42,43 Also, Mg2+ can poison the surface of HAp by adsorbing into the active growth sites and preventing further HAp nucleation.42 Thus, the removal of these ions should promote HAp formation, but ferulic acid is still being released, which has been shown to cause MCPM deposition. However, the nucleation process in mSBF occurs at a faster rate than in SBF because within 24 h in mSBF macroscopic deposits were observed on the surface polymers 2−4 (Figure 10), whereas microscopic deposits were observed

Figure 9. TGA evaluation of polymer 2 before and after mineralization for 2 weeks. Overlaid with MCPM (as received).

highest observed increase in Td, and it is consistent with polymer 2 gaining the most weight (Chart 1). This effect has been reported in the literature and is proposed to be due to calcium chelation to the polymer.35 (2) Two new weight loss inflections were found: 5% weight loss to 91 °C and 3% weight loss to 211 °C. These correspond to two endothermic Tm’s at 71 and 245 °C, which are due to partial hydrate losses from the hydrate in MCPM. (3) An increase occurred in the amount of sample residue after 800 °C (4.6% for polymer 2). This was further evidence of the presence of calcium phosphate. As a reference, the as-received MCPM is overlaid in Figure 9. Four distinct transitions exist; however, these transitions vary depending on which solution MCPM is crystallized from.29 It is assumed that the more weight the mineralized samples gain, the closer the TGA curve will resemble that of MCPM. Mechanism of Apatite Nucleation. By combining all of the evidence obtained by ESEM, XRD, FTIR, and DSC/TGA, it was established that MCPM was nucleated from SBF and HAp was nucleated from mSBF. The mechanism of apatite nucleation from an SBF solution begins with calcium ions coordinating to the ionic functional groups. This mechanism was confirmed by the lack of deposition from the NaCl solution and the deposition from mSBF. However, different phases are produced from SBF (MCPM) and mSBF (HAp). On the basis of theoretical models, the nucleation of octacalcium phosphate (OCP) is preferred over HAp at physiological pH. The formation of HAp is only preferred at higher pH’s. However, it is the most thermodynamically favored state.36 MCPM is an acidic biological apatite that is typically only precipitated from acidic solutions.37 It is used as a precursor for bone cements because, when mixed with basic tricalcium phosphate in water, it forms an injectable paste.38 Therefore, OCP should nucleate from both solutions, but it is not nucleated in either. Thus, two questions need to be answered: (1) why does MCPM nucleate at all? (2) Why does MCPM precipitate from SBF but HAp precipitate from mSBF? MCPM is known to precipitate from an acidic solution,37 but studies have shown that it will also precipitate from an SBF solution (pH 7.4) in the presence of acidic anions, such as citrate.39 Citrate anions inhibit the growth of HAp because they replace the phosphate anions in the crystal structure of HAp.40 Only at concentrations of 0.3−2.0 mM citrate was the formation of HAp favorable. Above or below those concentrations, no HAp was formed;41 only MCPM/DCPD

Figure 10. Image of polymer 3 exposed to mSBF after 1 week.

in SBF. Thus, HAp was able to nucleate before enough ferulic acid was released to suppress the HAp formation. Once a HAp seed was produced, this promoted further HAp deposition.

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CONCLUSIONS A series of ferulic acid containing polyphosphazenes were synthesized and evaluated for their prospective bioactivity upon exposure to solutions of SBF. Several polymers were synthesized, and their side-group chemistry was modified to generate hydrophobic and hydrophilic polymers. Exposure to traditional SBF resulted in the nucleation of MCPM, a type of biological apatite frequently used in bone cements. Furthermore, there was a distinct dependence on the hydrophilicity of the polymers. Hydrophilic polymers (2 and 3) nucleated the 3508

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(14) Allcock, H. R. Chemistry and Applications of Polyphosphazenes; John Wiley and Sons: Hoboken, NJ, 2003. (15) Allcock, H. R.; Morozowich, N. L. Polym. Chem. 2012, 3, 578− 590. (16) Deng, M.; Nair, L. S.; Nukavarapu, S. P.; Kumbar, S. G.; Jiang, T.; Krogman, N. R.; Singh, A.; Allcock, H. R.; Laurencin, C. T. Biomaterials 2008, 29, 337−349. (17) Krogman, N. R.; Weikel, A. L.; Kristhart, K. A.; Nukavarapu, S. P.; Deng, M.; Nair, L. S.; Laurencin, C. T.; Allcock, H. R. Biomaterials 2009, 30, 3035−3041. (18) Sethuranman, S.; Nair, L. S.; El-Amin, S.; Farrar, R.; Nguyen, N. M.-T.; Singh, A.; Allcock, H. R.; Greish, Y. E.; Brown, P. W.; Laurencin, C. T. J. Biomed. Mater. Res. A 2006, 77, 679−687. (19) Weikel, A. L.; Owens, S. G.; Morozowich, N. L.; Deng, M.; Nair, L. S.; Laurencin, C. T.; Allcock, H. R. Biomaterials 2010, 31, 8507− 8515. (20) Deng, M.; Nair, L. S.; Nukavarapu, S. P.; Jing, T.; Kanner, W. A.; Li, X.; Kumbar, S. G.; Weikel, A. L.; Krogman, N. R.; Allcock, H. R.; Laurencin, C. T. Biomaterials 2010, 31, 4898−4908. (21) Morozowich, N. L.; Nichol, J. L.; Mondschein, R. J.; Allcock, H. R. Polym. Chem. 2012, 3, 778−786. (22) Sheweita, S. A.; Khoshhal, K. I. Curr. Drug Metab. 2007, 8 (5), 519−525. (23) Wattamwar, P. P.; Hardas, S. S.; Butterfiled, D. A.; Anderson, K. W.; Dziubla, T. D. J. Biomed. Mater. Res. A 2011, 99, 184−191. (24) Pangborn, A.; Giardello, M.; Grubbs, R.; Rosen, R.; Timmers, F. Organometallics 1996, 15, 1518. (25) Allcock, H. R.; Kugel, R. L. J. Am. Chem. Soc. 1965, 87, 4216. (26) Kokubo, T.; Kushitani, H.; Sakka, S.; Kitsugi, T.; Yamamuro, T. J. Biomed. Mater. Res. 1990, 24, 721−734. (27) Chun, C.; Lee, S. M.; Kim, C. W.; Hong, K.-Y.; Kim, S. Y.; Yang, H. K.; Song, S.-C. Biomaterials 2009, 30, 4752−4762. (28) Allcock, H. R.; McDonnell, G. S.; Riding, G. H.; Manners, I. Chem. Mater. 1990, 2, 425−432. (29) Boonchom, B.; Danvirutai, C. J. Opt. Biomed. Mater. 2009, 1 (1), 115−123. (30) Posner, A. S. Physiol. Rev. 1969, 49, 760−792. (31) Silverstein, R. M.; Webster, F. X. Infarared Spectroscopy. Spectrometric Identification of Organic Compounds, 6th ed.; John Wiley & Sons, Inc.: New York, 1998; pp 96−97. (32) Xu, J.; Gilson, S. F. R.; Butler, I. S. Spectrochim. Acta, Part A 1995, 54, 1869−1878. (33) Janaki, K.; Elamathi, S.; Sangeetha, D. Trends Biomater. Artif. Organs 2008, 22 (3), 169−178. (34) Elliott, J. C. Structure and Chemistry of the Apatite and Other Calcium Orthophosphates; Elsevier: Amsterdam, The Netherlands, 1994. (35) Manjubala, I.; Scheler, S.; Bossert, J.; Jandt, K. D. Acta Biomater. 2006, 2, 75−84. (36) Lu, X.; Leng, Y. Biomaterials 2005, 26, 1097−1108. (37) Dorozhkin, S. V.; Epple, M. Angew. Chem., Int. Ed. 2002, 41, 3130−3146. (38) Huan, Z.; Jiang Chang, J. Acta Biomater. 2009, 5, 1253−1264. (39) Zainuddin, Hill, D. J. T.; Whittaker, A. K.; Kemp, A.; Chirila, T. V. The role of citrate anions in prevention of calcification of poly(2hydroxyethyl methylacrylate) hydrogels. In Polymer Durability and Radiation Effects; Celina, M., Ed.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008; Chapter 25. (40) Mirsa, D. N. J. Dent. Res. 1996, 75, 1418−1425. (41) Rhee, S.-H.; Tanaka, J. Biomaterials 1999, 20, 2155−2160. (42) Abbona, F. J. Cryst. Growth 1990, 104, 661−671. (43) LeGeros, R. Z.; Kijkowska, R.; Bautista, C.; LeGeros, J. P. Connect. Tissue Res. 1995, 33, 203−209.

most MCPM in the shortest period of time (within 1 week), whereas hydrophobic polymers (6 and 7) had very slow deposition times (∼3 weeks). Polymers 2 and 3 gained 19− 27% after 4 weeks, polymers 4 and 5 gained 14−17%, and polymers 6 and 7 had little to no weight gain. The results collected by ESEM, XRD, FTIR, and TGA/DSC confirm the bioactivity of polymers 2−5. Subsequently, the method of nucleation was investigated by exposure of polymers 2−5 to standard solutions. Exposure to a NaCl solution left the polymers unchanged. Exposure to mSBF, which was free of crystal growth inhibitors Mg2+ and HCO3−, nucleated HAp. The deposition of HAp from mSBF was approximately two or three times faster than that of MCPM from SBF, with polymers 2−5 gaining 13−43% after only 2 weeks. Ideally, it is preferred for polymers to nucleate HAp from traditional SBF; however, the ability for polymers 2−5 to form biologically relevant apatite coatings within a short period of time may increase the osteoconductivity and osteoinductivity of the polymers, making them excellent candidates for implantable bone grafts.

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ASSOCIATED CONTENT

S Supporting Information *

Characterization data for polymers 2−5 and weight loss data for polymers 2−7. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Dr. Josh Stapleton for his assistance with the FTIR and meaningful discussions and Nichole Wonderling for the XRD and program analysis training. This publication was supported by The Pennsylvania State University Materials Research Institute Nanofabrication Lab and National Science Foundation Cooperative Agreement ECS-0335765.

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REFERENCES

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