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A biodegradable thermoset polymer made by esterification of citric acid and glycerol
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  A biodegradable thermoset polymer made by esterification of citricacid and glycerol Jeffrey M. Halpern, 1 Richard Urbanski, 2 Allison K. Weinstock, 2 David F. Iwig, 3 Robert T. Mathers, 2 Horst A. von Recum 1 1 Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio 44106 2 Department of Chemistry, Pennsylvania State University, New Kensington, Pennsylvania 15068 3 Alcoa Technical Center, 100 Technical Drive, Alcoa Center, Pennsylvania 15069Received 15 February 2013; revised 21 May 2013; accepted 22 May 2013Published online 24 June 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.34821 Abstract:  A new biomaterial, a degradable thermoset poly-mer, was made from simple, economical, biocompatablemonomers without the need for a catalyst. Glycerol and citricacid, nontoxic and renewable reagents, were crosslinked by amelt polymerization reaction at temperatures from 90 to150  C. Consistent with a condensation reaction, water wasdetermined to be the primary byproduct. The amount of crosslinking was controlled by the reaction conditions,including temperature, reaction time, and ratio between glyc-erol and citric acid. Also, the amount of crosslinking wasinversely proportional to the rate of degradation. As a proof-of-principle for drug delivery applications, gentamicin, anantibiotic, was incorporated into the polymer with prelimi-nary evaluations of antimicrobial activity. The polymersincorporating gentamicin had significantly better bacteriaclearing of   Staphylococcus aureus   compared to non-gentamicin gels for up to 9 days.  V C 2013 Wiley Periodicals, Inc.J Biomed Mater Res Part A: 102A: 1467–1477, 2014. Key Words:  controlled drug release, glycerol, polyester, citricacid, degradable polymers How to cite this article:  Halpern JM, Urbanski R, Weinstock AK, Iwig DF, Mathers RT, von Recum HA. 2014. A biodegradablethermoset polymer made by esterification of citric acid and glycerol. J Biomed Mater Res Part A 2014:102A:1467–1477. INTRODUCTION The use of crosslinking in polymers, effectively generatingthermoset materials, has received widespread attention as ameans to tailor device properties for use in vascular andosseous tissue. 1–4 The improved mechanical performance of crosslinked biomaterials aids as a scaffold for cell growth,as well as varying degrees of controlled drug release, orbiodegradability. 2,5,6 Biodegradable polymers in biomedical applications arefrequently made with ester bonds, due to their capacity forhydrolytic cleavage, although other linkages based on car-bonyl derivatives, such as imines, amides, and anhydrides,have also been reported. 6–10 Such clinically used polyestersare typically thermoplastic polymers, such as poly(lacticacid) (PLA), poly( e -caprolactone), and poly( L -lactic- co -gly-colic acid) (PLGA). 11,12 In contrast to thermoset polymers,thermoplastic polymers are less mechanically robust, whichlimits the ability to tailor them for a broad range of applications.Various biodegradable polymers, made from either citricacid or glycerol, have previously been researched withmixed results. Biodegradable ester materials based on glyc-erol were made with various carboxylates (e.g., sebacate),fabricated at a high temperature under a low-pressureargon environment. 13–15 For example, glycerol and sebacicacid were reacted without a catalyst to form poly-glycerolsebacate (PGS), which shows promising biocompatibilityand biodegradability results. 15 However, due to the highhydrophobicity of sebacic acid, degradation and biologicalfate of the monomers and short oligomers are often compli-cated by solubility issues. The local release of sebacic acidleads to a higher concern about local pH change than wouldoccur upon release of hydrophilic citric acid. Further, citricacid, as used herein, is more readily available and of lowercost than sebacic acid. In regards to polymers based on cit-ric acid, other papers have described reacting citric acidwith polyethylene glycol to create thermoplastic tri-block dendrimer macromolecules and nanomolecules for drugdelivery systems; however, these polymers showed limitedbiodegradability. 16–19 Also the thermoplastic materialswould have minimal branching, and therefore only modest mechanical property change, as compared to the high cross-linking potential of the described thermoset materials.Finally, citric acid was previously reacted with glycerol insolution and in the presence of benzene and p-toluenesulfonic acid (PTSA) to form a crosslinked estercopolymer. 20 Although this resulting citric acid and glycerolpolyester showed promise as a drug delivery system, the Correspondence to:  H. A. von Recum; e-mail: hav1@case.eduContract grant sponsor: National Institutes of Health Ruth L. Kirschstein National Research Service Award; contract grant number: T32 AR007505 V C 2013 WILEY PERIODICALS, INC. 1467  incorporation of carcinogens, such as benzene and PTSA,created compatibility complications for biomedical andpharmaceutical applications.Our research group has explored the synthesis of a newcrosslinked, thermoset polymer, which can be made with awide range of degradation and mechanical parameters andis made from simple, economical, bio-available reagentswithout the need for a catalyst. In addition, the chemistry isnoncomplex and can be conducted in air, at atmosphericpressure. Our goal was to design a polymer with the follow-ing properties: (a) uses ester bonds to take advantage of hydrolytic cleavage; (b) is only made from low cost, non-toxic renewable components; (c) retains the capacity to con-trol the rate of degradation; (d) has the capability of incorporating chemical functionalities, deliverable drugs,and nutrients. For the first two properties, the use of non-toxic, pharmaceutical grade, ester bond-forming compo-nents, we identified citric acid and glycerol (listed as one of top 12 renewable chemicals by the United States Depart-ment of Energy) 21 as nontoxic renewable resources and bio-logically safe nutrients, being generally regarded as safe(GRAS) by the United States Food and Drug Administra-tion. 4,22,23 Both have been identified as building blocks for aplatform to deliver pharmaceuticals. 16–20,24,25 For the thirddesired property, the rate of degradation has been found tobe inversely proportional to the amount of crosslinking, andit is possible to vary the amount of crosslinking of citricacid and glycerol. Finally, chemical functionality or deliver-able payload (e.g., antibiotics) can be integrated into thiscrosslinked system during fabrication to create an effectivedelivery mechanism.This article describes the esterification of citric acid andglycerol using a condensation reaction mechanism to fabri-cate a new thermoset polymer capable of drug delivery. Ini-tial studies used conventional catalysts; however, weobserved a high yield even without the use of catalysts.Varying the amount of glycerol was a convenient method tocontrol the physical properties, degree of crosslinking, andbiodegradability. Additionally, the melt polymerization onlyproduced water as a byproduct of the condensation reac-tion. In proof-of-principle studies, gentamicin was incorpo-rated into the polymer to serve as a model drug, as itspresence can be easily evaluated by its antibacterial proper-ties. Although ongoing work is underway in our labs toevaluate drug loading following polymer synthesis, or tosynthesize the polymer under lower temperatures, the cur-rent materials require drugs which are stable at moderatelyhigh temperatures. Since gentamicin is well-know to showminimal to no degradation at temperatures below 121  C, 26 is served an excellent model drug for incorporation anddelivery in the current system. EXPERIMENTAL Materials Glycerol (99%, Sigma-Aldrich),  para -toluenesulfonic acidmonohydrate (PTSA) (97.5%, Acros Organics), zinc (II) chlo-ride (97 1 %, Acros Organics), and gentamicin (BioReagent,Sigma-Aldrich) were used as received. Citric acid(anhydrous, 99.5%, Acros) was ground into powder withmortar and pestle and filtered through a brass sieve (150 m m, Fisher Scientific). Aluminum pans (7-cm diameter,Fisher Scientific) and buffer solution pH 7.40 (certified pH7.39–7.41 @ 25  C, Fisher Scientific) were used as received. General method for polymerization of glycerol andcitric acid Freshly ground anhydrous citric acid powder (8.0 mmol)was sieved through a 150  l m brass sieve into a 7.0 cm alu-minum pan with glycerol (8.0 mmol). One experimental con-trol dish was set up with no catalyst added. The two otherdishes also contained PTSA (0.08 mmol, 1 mol %) andZnCl 2  (0.08 mmol, 1 mol %). The three dishes were placedin oven set at a set temperature and time before beingremoved and allowed to cool to ambient temperature.Subsequent trials of polymerization methods include theincrease of the glycerol to citric acid ratio from 1:1 to 2:1and 3:1, as well as other variations specified within the text.For the gentamicin incorporated experiments, 1.6:1([glycerol]:[citric acid]) and 5 mol % gentamicin was poly-merized at 110  C for three different reaction times—7, 15,and 48 h—to create low crosslinked, medium crosslinked,and high crosslinked polymers, respectively. 5 mol % genta-micin was chosen because it successfully dissolved in glyc-erol, and also, this concentration lead toward effectiveantibacterial activity, however further optimization ispossible. Polymer characterization The decomposition temperature ( T  d ) was determined with aTA Instruments thermo-gravimetric analysis (TGA) Q500 at 20  C/min under a flow of nitrogen (30 mL/min). Polymersamples (4–8 mg) were placed on platinum pans andheated from 30 to 650  C. The reported decomposition tem-perature ( T  d ) values were calculated from the onset of decomposition using the peak from the first derivative of the weight loss to identify the maximum slope.Mechanical analysis was measured with a TA Instru-ments Q800 dynamic mechanical analyzer (DMA). A fivepoint temperature calibration was performed. The reactionof glycerol and citric acid ([gly]/[CA] 5 1) was examined at a frequency of 1 Hz and amplitude of 30  m m. The samplebar (35  3  13  3  1.7 mm 3 ), which was backed with alumi-num foil, was removed from a Teflon mold after curing for2 h and placed in a single cantilever clamp. The moduluswas measured at 110  C.FTIR spectra (32 scans) were recorded with a ZnSe ATR crystal at a 4 cm 2 1 resolution on a ThermoFisher Nicolet iS10 FTIR spectrometer. Kinetic evaluation of glycerol and citric acid Reaction kinetics were evaluated by measuring the waterlost in relation to the percentage of hydroxyl groupsreacted. Three separate trials of 1:1 ratio glycerol to citricacid were set up, and each was run in a gas chromatogra-phy (GC) oven at separate temperatures of 90, 110, and130  C for a minimum of 10 h each. Samples were removed 1468 HALPERN ET AL. BIODEGRADABLE THERMOSET POLYMER  from the oven at intervals and weighed. The weights wererecorded and calculated for weight loss to determine theformation of ester groups. Given the large boiling point dif-ferences between glycerol (bpt 290  C) and water at atmos-pheric pressure, all mass loss was attributed to waterformation. The % OH groups that reacted were calculatedwith the formula: (g of water lost)  3  (1/18.015 g mol 2 1 )  3 (1/maximum mol water)  3  100. The maximum mol of water that could be theoretically produced by esterificationreactions was: (g citric acid/192.12 g mol 2 1 )  3  3. Thekinetic profiles were obtained by graphing the resulting %OH values as a function of time. Liquid chromotography mass spectrospcopy characterization High performance liquid chromatography (HPLC)-gradewater was added to a resin sample and was thoroughlymixed. After 1 h, a 1  m L aliquot was injected into a WatersAcquity UPLC in line with a Thermo Scientific LTQ-Orbitrapin ESI( 1 ) mode. The UPLC system was equipped with aBEH phenyl column (130 Å, 1.7  m m, 2.1  3  75 mm 2 ) equili-brated in 95% solvent A (0.1% formic acid) and 5% solvent B (0.1% formic acid in acetonitrile) at 0.400 mL/min. Massspectra data were collected using full Fourier transformmode with 30,000 resolution. The compounds containinggentamicin eluted between 0.40 and 0.60 min; the massspectra across all peaks in this time period were averaged,and the neutral mass spectrum was extracted using theassociated Thermo Scientific Qual Browser 2.0.7 SP1software. Bacteria-clearing assays Antimicrobial activity was examined using both a dynamic,solution-based bacterial clearing assay and a static zone of inhibition study. The high agitation of the solution-basedassay tends to more effectively model mixing, solvent action,and removal of degradation products than a static assay.The zone of inhibition assay tends to have higher sensitivityand to better model the low vascularity and diffusionalexchange present in the environments in which many of these materials are used (e.g., subcutaneous, intraosseous),compared to a dynamic, solution-based assay. Although nei-ther are accurate predictors of biological performance, bothhave been previously used to describe new materials anddelivery systems and to indicate that the release is applica-tion dependent. 27–32 Similarly, the ASTM E2149–10 Stand-ard Test Method calls for both solution testing and zone of inhibition assays. 33 For the solution-based bacterial assay 30 g BBL Trypti-case Soy Broth (Becton, Dickinson Company) was dissolvedin 1 L Milli-Q water and autoclaved at 121  C for 20 min.Bacteria were freshly grown by placing frozen  Staphylococ-cus aureus   ( S. aureus   kindly provided by Dr. Edward Green-field, Case Western Reserve University) into a 15 mL Falcontube prepared with 5 mL sterile soy broth and incubatedfor overnight at 37  C on an orbital shaker (  225 rpm).105–140 g of high, medium, and low crosslinking poly-mer were each placed into a 15-mL Falcon tube with 5 mLsoy broth. Two controls were prepared, (1) a tube with con-trol polymer not loaded with drug, and (2) a tube with nopolymer present, with the latter used to normalize themeasurements. Each tube was infected with 10  l L freshlygrown bacteria and incubated for 20–24 h on a 37  C orbitalshaker. The  S. aureus   solution was completely removed andreplaced with fresh  S. aureus   solution each day for a periodof 6 days. All tubes were done in triplicate.Each sample was prepared in three dilutions to ensureat least one measurement was in the linear range of the cal-ibration. The calibration curve was generated by producinga dilution series from the sample with no polymer. The sam-ples were read at two absorbance wavelengths, 485 and595 nm, and the determined % clearing was averaged,assuming that the control tubes with no polymer had 100%bacteria.For the static, zone of inhibiton assay, also known as theKirby-Bauer Assay, plates were prepared as previouslydescribed. 34 As mechanical breakdown and sample fragmen-tation occurred during the course of the study, it was chal-lenging to continue to transfer entire samples from plate toplate which is required for a conventional Kirby-BauerAssay. To circumvent this, samples were placed into aporous tissue culture insert with 1  m m pore size, and theassay run by moving this insert from plate to plate. Thisensured that released drug could escape to have antimicro-bial effect, but that as the sample fragmented all fragmentslarger than 1  m m were contained together. We had previ-ously validated this procedure using other work in our lab(manuscript under review). In this study, specifically 32 mgof medium and high crosslinked polymer were placed inTranswell porous tissue culture inserts (6 cm) ( N  5 3).Before the zone of inhibition assay, the samples weresoaked for 1.5 h in a 200  l L phosphate buffered saline(PBS) solution. The water was removed by Kimwipe under-neath the Transwell. In addition, 20  l L of PBS solution wasadded after every transfer to aid in media transfer betweenpolymer and bacteria-infected soy broth agar. The zoneswere measured and the Transwell plates were transferredto new bacteria plates every day. RESULTS Polymer formation and characterization The reaction of an alcohol with a carboxylic acid is a well-studied reaction that forms an ester under noncatalytic orcatalytic conditions. 35 Common catalysts include Bronstedacids, Lewis acids, enzymes, and solid acids. As demon-strated by TGA data in Figure 1, the melt polymerization of glycerol and citric acid with catalysts PTSA and ZnCl 2  gener-ates a polyester network with greater thermal stability com-pared to the onset of weight loss for citric acid (197  C) andglycerol (209  C). In addition, characterization of a samplebar by dynamic mechanical analysis (DMA) indicated anincrease in the storage modulus (Fig. 2) as a function of time. The large increase in the storage modulus indicatedthat the reaction of glycerol and citric acid produced acrosslinked network with robust physical properties. DMAalso detected glass transition temperatures ( T  g ) based on ORIGINAL ARTICLE JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A  | MAY 2014 VOL 102A, ISSUE 5 1469  the maximum of the tan  d  peak, which represents the ratioof storage to loss moduli. After heating the film at 110  C,the  T  g  increased to 61  C after 24 h, and the  T  g  increased to83  C after 48 h. These data demonstrate the generation of acrosslinked network between citric acid and glycerol.In the absence of a catalyst, the equilibrium could beshifted toward the products by the removal of water, eitherby increasing the temperature or by decreasing the pres-sure. As depicted in Scheme 1, the reaction between glyc-erol and citric acid proceeded at temperatures above 90  C.Using FTIR spectroscopy, the ester formation was accompa-nied by a decrease in the OH (3290 cm 2 1 ) and C A O (1032cm 2 1 ) absorbances for glycerol. In Figure 3, the initial C @ Oabsorbance for citric acid (1694 cm 2 1 ) was graduallyreplaced by ester absorbances at 1724 cm 2 1 (C @ O stretch)and 1176 cm 2 1 (C A O).In order to determine the optimum ratio of [glycerol]/[citric acid] for fabricating a polymer useable in drug deliv-ery, several ratios were characterized by TGA (Fig. 4). Thetwo-stage decomposition profile of the TGA curves indicatesthat the crosslinking between glycerol and citric aciddepends on the molar ratio of [glycerol]:[citric acid]. The FIGURE 1 . TGA data (20  C/min) for the reaction of glycerol and citricacid at 110  C using 1 mol % paratoluenesulfonic acid, 1 mol % ZnCl 2 ,and no catalyst. TGA data for unreacted citric acid is shown for com-parison. Top: Weight percent as a function of temperature. Bottom:Derivative of weight percent as a function of temperature. FIGURE 2 . Dynamic mechanical analysis (DMA) of the reaction of glycerol and citric acid ([glycerol]:[citric acid] 5 1) at 110  C. A samplebar (35  3  13  3  1.7 mm 3 ) was oscillated at 1 Hz in a single cantileverclamp using a 30-m amplitude. SCHEME 1 . The synthesis of a polyester network using glycerol and citric acid. The dashed lines represent additional network connections. 1470 HALPERN ET AL. BIODEGRADABLE THERMOSET POLYMER  samples with a 1:1 glycerol to citric acid ratio had 30%decomposition at 300  C compared to the samples with 2:1and 3:1 ratios, which had 60% decomposition. This compar-ison indicates that the optimum ratio of [glycerol]:[citricacid] for biomedical applications will be expected to fallbetween 1:1 and 2:1.In addition to controlling the degree of crosslinking withthe ratio of [glycerol]/[citric acid], the influence of time onthe polyester thermoset was also investigated. In Figure 5,samples with a 1:1 ratio of glycerol to citric acid were pre-pared and reacted at 150  C for 0.5, 1.0, and 3.0 h. All threesamples in this experiment showed two different stages of weight loss that was previously seen in Figure 1. As timewas increased, an increase in the amount of ester formationwas observed. After a 3.0 h reaction time, a higher percent-age of polymer remained at 325  C with a concomitant decrease in the initial decomposition of the sample. As it was observed that the reactivity at 150  C proceeded veryquickly, even after 0.5 h without a catalyst, lower tempera-tures were examined to investigate the optimal method forthe synthesis of a drug delivery system. Kinetic data of crosslinked reaction In Figure 6, the kinetic data from the reaction of glyceroland citric acid was examined by measuring the percent of OH groups reacted as a function of time. As this reactionundergoes Fischer esterification, producing water as abyproduct, the percent of OH groups reacted was calculatedby measuring the proportional amount of water loss. The FIGURE 3 . FTIR spectroscopy data for the reaction of glycerol and cit-ric acid ([glycerol]:[citric acid] 5 1.4) at 110  C showing the carbonylregion for citric acid, product after 10 min, and product after 150 minas the ester absorbance at 1724 cm 2 1 becomes more pronounced. FIGURE 4 . Overlay of TGA data (20  C/min) for reaction of glycerol andcitric acid at 150  C for 1 h using [glycerol]:[citric acid] ratios of 1:1,2:1, and 3:1. Top: Weight percent as a function of temperature. Bot-tom: Derivative of weight percent as a function of temperature. FIGURE 5 . TGA data (20  C/min) showing the influence of time on thereaction of glycerol and citric acid of a 1:1 ratio at 150  C for 0.5, 1.0,and 3.0 h. Top: Weight percent as a function of temperature. Bottom:Derivative of weight percent as a function of temperature. ORIGINAL ARTICLE JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A  | MAY 2014 VOL 102A, ISSUE 5 1471
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