European Polymer Journal 46 (2010) 663–672

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European Polymer Journal

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Preparation of macroporous monoliths based on epoxy-bearing hydrophilicterpolymers and applied for affinity separations

Ruben Dario Arrua a, Cristian Moya b, Eugenia Bernardi b, Jorge Zarzur b, Miriam Strumia a,Cecilia I. Alvarez Igarzabal a,*

a Departamento de Química Orgánica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba. Ciudad Universitaria, Córdoba 5000, Argentinab Laboratorio de Hemoderivados, Universidad Nacional de Córdoba, Córdoba, Argentina

a r t i c l e i n f o

Article history:Received 13 August 2009Received in revised form 30 December 2009Accepted 11 January 2010Available online 15 January 2010

Keywords:Macroporous monolithsMacroporous polymersChemical modificationHeparin immobilization

0014-3057/$ - see front matter � 2010 Elsevier Ltddoi:10.1016/j.eurpolymj.2010.01.009

* Corresponding author. Fax: +54 0351 4333030/E-mail address: cia@mail.fcq.unc.edu.ar (Cecilia

a b s t r a c t

This work reports the synthesis and characterization of poly(N-acryloyl-tris(hydroxy-methyl)aminomethane-co-glycidyl methacrylate-co-N,N0-methylenebisacrylamide) [poly-(NAT-GMA-BIS)] discs using different porogenic solvents. The macroporous polymerobtained with PEG 6000 as co-porogen showed the best porous properties of the series.End-point immobilization of heparin (Hep, as ligand) was then carried out on the productsthrough reductive amination. Thus, the products were reacted with ethylenediamine (EDA)and hexamethylenediamine (HMDA) in order to introduce amine groups and to analyze thelength of the spacer on the immobilization of heparin. Then, the highest value of aminegroups [1.280 and 0.919 mmol amine/g dry support for poly(NAT-GMA-BIS)-EDA andpoly(NAT-GMA-BIS)-HMDA, respectively] was obtained at 40 �C after 48 h of reaction.Finally, the amount of ligand coupled to discs was not influenced by the two length spacersassayed. The amount of Hep coupled on discs [591.50 and 489.90 lg Hep/g dry polymer forpoly(NAT-GMA-BIS)-EDA-Hep and poly(NAT-GMA-BIS)-HMDA-Hep, respectively] wassimilar to that obtained using macroporous polymer beads reported by other authors.The supports yielded were used to assay the retention of antithrombin III (AT-III). Theretention was greater for the EDA-containing product with a larger amount of heparin.These novel heparin-containing macroporous poly(NAT-GMA-BIS) discs could be used aspotential affinity chromatography supports.

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1. Introduction

In the last years, macroporous monoliths have beenintroduced as a new and useful generation of macroporousmaterials. Each monolith consists of a permeable mass sin-gle-piece that can be compared with a simple and largeporous particle forming a continuous rod. Within themonolith, a series of connected pores creates a continuousskeleton, filled with interconnected pores that form flowchannels of a consistent size [1,2] allowing for high perme-ability and therefore flow rates at moderate pressures [3].

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4170/4173x151.I. Alvarez Igarzabal).

The macroporous polymers monoliths can be preparedin a simple way, from a homogenous mixture formed byvinylic/di-vinylic monomers and the inert or a mix of inertsolvents, into a mold to obtain finally a polymeric material.The accepted mechanism of pore formation in a polymeri-zation between mono-vinyl monomers and di-vinyl mono-mers, initiator and porogenic solvent, is similar to thatused for a polymer bead and can be summarized as follow:the formed radicals start the reaction in solution afterwhich the polymers precipitate when become insolublein the reaction medium. The nuclei swell with the mono-mers present in the surrounding liquid, and the polymeri-zation continues in solution but preferably within theswollen nuclei where the local concentration of the mono-mers is higher in them because the monomers are thermo-

664 R.D. Arrua et al. / European Polymer Journal 46 (2010) 663–672

dynamically better solvating agent for the polymer thanthe porogen. If the monomers are solid and were dissolvedto react, they probably tend to leave a less polar medium inorder to stay within the more polar swollen nuclei, whichlead to a higher local concentration of monomers in theswollen nuclei than in the surrounding solution. The poly-mer chains in formation are captured by the growing nu-clei and further increase their sizes. The nuclei associatein clusters being held together by polymer chains thatcrosslink the neighboring nuclei. So, the interconnectedand grown clusters form a final porous matrix within thepolymerization system [4]. For these syntheses, differenttype of reactors can be used as tubes [3,5,6], disks [7], orcylinders [8]. All these polymeric systems were obtainedunder careful studies of the reaction parameters that influ-ence the porous architecture of the monoliths yielded.

The high flow rates with the convective mass transfer,possibilities shorter separations times when these materialsare used as stationary phase in chromatographic separa-tions. Due to their porous characteristics, they can beobviously used in different chromatography processes suchas ion exchange chromatography [9], as stationary phasesfor HPLC [10–16] and affinity chromatography [17–22], orused in capillary electrochromatography [23–24] or inmicrofluidic chips technology [25]. The more commonmonolithic columns for larger molecules (proteins, nucleicacids, synthetic polymers) are based on organic polymers,although silica based-supports are widely used in separa-tions of small molecules. Besides, depending on the mono-mers used in the synthesis, macroporous polymersmonoliths with different surface chemistry are obtained.Thus, those based on hydrophilic acrylates [4,6,26] andacrylamide monomers [12,27] constitutes hydrophilic poly-mer rods while those formed from different styrene-basedmonomers [28] are non-polar monoliths. Nevertheless, theepoxy groups-containing monoliths as those based in glyc-idyl methacrylate are very useful, since these functionalgroups present in these materials can be easily reacted withspecific ligands to yield an affinity chromatography support.In general, the monoliths for chromatography have proper-ties as fast operation (separation in very short period oftime) and higher capacity and efficiency for large biomole-cules. For the application of affinity chromatography inporous media, the covalent binding of a ligand onto the poly-meric material is necessary. The surface functionalizationand chemistry and porous properties of the monoliths canbe tailored adjusting the composition of the initial monomersolution and the polymerization conditions [25].

Recently, we have studied the influence of various reac-tion parameters on the porous properties of hydrophilicpoly(N-acryloyl-tris(hydroxymethyl)aminomethane-co-gly-cidyl methacrylate-co-N,N0-methylenebisacrylamide) [poly-(NAT-GMA-BIS)] macroporous discs [29]. These polymerscontain NAT and GMA monomers. NAT, with hydroxyl-methyl triads attached to it as side groups confers hydrophi-licity to the surface polymer, whereas GMA offers epoxygroups through which further modification reactions overthe surface can be performed. Fig. 1 shows the molecularstructures of the three monomers used.

The present work reports the preparation of macropo-rous poly(NAT-GMA-BIS) discs using different series of

porogenic solvents. These, constituted new polymer sta-tionary phases of augmented hydrophilicity for biosepara-tions. So, after to realize the end-point immobilization ofheparin as ligand on discs, the final products were usedto retain antithrombin III (AT-III) to demonstrate their po-tential application as affinity chromatography supports.

Heparin is a sulfated polysaccharide belonging to thefamily of glycosaminoglycans [30]. Due to its polyanionicnature and carbohydrate sequence, heparin is able to inter-act with proteins such as blood-clotting factors and humanserum lipoproteins. The immobilization of heparin on dif-ferent chromatography supports was thus developed forthe purification of proteins and other biopolymers [31–36].

In the preparation of affinity supports, the importanceof the mode of attachment of the ligand to the matrix iswell known. Hence, it is essential that once the ligandhas been immobilized on the matrix, the active sites in-volved in the specific interaction with the biomolecule tobe retained remain accessible. Therefore, the immobiliza-tion of heparin was carried out with different methods,such as CNBr-activated Sepharose [37] and amino or car-boxyl functionalized supports [38]. Nevertheless, better re-sults were obtained when heparin was bound to amino-functionalized supports through reductive amination,involving the aldehyde group at the reducing end of theheparin molecule (end-point immobilization). Throughthis synthetic route, heparin was bound to soft gel materi-als such as Sepharose [38], although these biopolymersshowed low mechanical properties to be used in liquidchromatography systems in which fast flow rates and rel-atively low backpressures are required. Therefore, heparinwas immobilized by this method on silica-based rigidmaterials with improved mechanical properties [39].Thissame methodology was selected in this work to performthe immobilization of heparin on amino-modified poly(NAT-GMA-BIS) discs.

2. Experimental

2.1. Materials

N-Acryloyl-tris(hydroxymethyl)aminomethane (NAT)(Aldrich, Steinheim, Germany); N,N0-methylenebisacryla-mide (BIS) (Mallinckrodt, Kentucky, USA); glycidylmethacrylate (GMA) (Aldrich, Steinheim, Germany); azobisi-sobutyronitrile (AIBN) (Aldrich, Steinheim, Germany);dimethylsulfoxide (DMSO) (Anedra, Argentina); ethanol(Cicarelli, Argentina); n-pentanol (BDH, England); n-octanol(Rield De Haen, Europe); n-dodecanol (Merck, Hohenbrunn,Germany); n-tetradecanol (Fluka, Steinheim, Germany);polyethylenglycol 6000 (PEG 6000) (Fluka, Steinheim,Germany); ethylenediamine (EDA) (Carlo Erba, France); hex-amethylenediamine, 98% (HMDA) (Aldrich, Steinheim,Germany); ethanolamine (Aldrich, Steinheim, Germany);heparin, sodium salt (Hep) (Sigma, St. Louis, USA); sodiumcyanoborohydride, 94% (NaBH3CN) (Sigma, St. Louis, USA);acetic anhydride (Merck, Germany); trinitrobenzenesul-phonic acid (TNBS), 1 M in water (Fluka, Steinheim,Germany); sodium borate (Anedra, Argentina); picrylsulfonicacid (Fluka, Steinheim, Germany); toluidine blue (Aldrich,

Fig. 1. Molecular structures of N-acryloyl-tris(hydroxymethyl)aminomethane (a), glycidyl methacrylate (b) and N,N0-methylenebisacrylamide (c)monomers used in the synthesis of macroporous polymers.

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Steinheim, Germany) and antithrombin III (AT-III) (providedby Laboratorio de Hemoderivados, Universidad Nacional deCórdoba) were used as received.

2.2. Preparation of poly(NAT-GMA-BIS) macroporous discs

Since NAT and BIS are solid, their dissolution in the por-ogenic mixtures was carried out in glass tubes at the reac-tion temperature. Thus, mono-vinyl monomers (0.7357 gof NAT and 0.57 mL of GMA) and the cross-linker(0.8633 g of BIS) were dissolved in a porogenic mixtureof 2.88 mL of DMSO and 1.12 mL of different porogenagents [ethanol, n-pentanol, n-octanol, n-dodecanol, n-tet-radecanol or poly(ethylenglycol) 6000 (PEG 6000)]. As ithas been dissolved at the reaction temperature, a homoge-neous reaction mixture was attained. After the dissolutionof monomers, the mixture was purged with nitrogen for10 min and AIBN was then added (1 wt.% with respect tomonomers). After that, the polymerization mixture wasplaced into polypropylene syringes for 24 h at 70 �C. Themonoliths were removed from the syringes and cut in3-mm thick discs using a lathe. Polymers were purifiedwith methanol in a Soxhlet for 24 h to eliminate the un-re-acted reagents, and dried under vacuum to constantweight to analyze their porous properties. Their epoxyequivalents were also determined by using the pyridiniumchloride method [40]. The products are shown in Table 1.

2.3. Porous properties in dry state

The pore size distribution of the monolithic materialswas determined by mercury intrusion porosimetry usingan Autopore II 9220 Micrometrics (Norcross, USA), and their

Table 1Polymerization conditions and porous properties of macroporous poly(NAT-GMA-

Polymera Co-porogen VpT (mL/g)b

Poly(NAT-GMA-BIS)-1 C2H5OH 0.044Poly(NAT-GMA-BIS)-2 C5H11OH 0.063Poly(NAT-GMA-BIS)-3 C8H17OH 0.076Poly(NAT-GMA-BIS)-4 C12H25OH 0.502Poly(NAT-GMA-BIS)-5 C14H29OH 0.661Poly(NAT-GMA-BIS)-6 PEG 6000 0.812

a Polymerization conditions: total mol monomers: 0.014 in a NAT:GMA:BIS relpolymerization time: 24 h; temperature: 70 �C; DMSO:co-porogen (% vol): 72:28

b Total pore volume.c Pore size at the highest peak in the pore size distribution profile.d Total specific surface area.e Porosity.

superficial morphologies were studied by EnvironmentalScanning Electron Microscopy (ESEM) using Philips XL-30TMP PW 6635/45 equipment (Eindhoven, Netherlands).The images were recorded with magnifications of 20,000�.

2.4. Derivatization reactions

2.4.1. Amination reactionsThese reactions were carried out under different condi-

tions using poly(NAT-GMA-BIS)-6 (Table 1) as matrix andethylenediamine (EDA) as a spacer to obtain poly(NAT-GMA-BIS)-EDA discs. Therefore, macroporous poly(NAT-GMA-BIS) discs (200 mg) were immersed in 10 mL ofdistilled water (twice) and then in 10 mL of 0.5 M phosphatebuffer pH 8.00 (twice) for 1 h each wash to equilibrate them.The discs were then transferred to beakers containing 6 mLof EDA solutions (0.057–0.573 g/mL) in the same buffer. Theamination reactions were allowed to proceed at a selectedtemperature (25 or 40 �C) for (8–264 and 2–48 h, respec-tively). Table 2 summarizes the results of these assays. Onceeach reaction had finished, the amine-containing discs werewashed with the buffer used in the reaction until no posi-tively TNBS test was observed from the effluents. To blockthe remaining epoxide groups, each disc was placed over-night in 6 mL 1 M ethanolamine solution in the same phos-phate buffer at room temperature. When the reaction wascomplete, the discs were washed first with the same buffer,until a negative TNBS test was observed from the effluents,and then with distilled water. The supports were stored at+4 �C in ethanol solution 20% vol. The best conditions forthe attachment of EDA (40 �C and 48 h) were used to bindhexamethylenediamine (HMDA) to yield poly(NAT-GMA-BIS)-HMDA-1 disc.

BIS) discs obtained using different co-porogen agents.

Dp (nm)c SsT (m2/g)d P (%)e

– 19.8 5.7– 26.0 7.8– 30.9 8.3

30.5 74.5 40.472.4 58.3 47.9

280.7 15.4 51.5

ation of 30:30:40, respectively; AIBN: 1% w/w with respect to monomers;with respect to total volume of the porogenic mixture equal to 4 mL.

Table 2Experimental reaction conditions (time and temperature) on poly(NAT-GMA-BIS)-6 to yield aminated discs.

Polymera Time(h)

Temp.(�C)

Amount ofamine groups(mmolamine/g drypolymer)b

Yield(%)c

Poly(NAT-GMA-BIS)-EDA-1 8 25 0.527 42.50Poly(NAT-GMA-BIS)-EDA-2 15 25 0.522 42.10Poly(NAT-GMA-BIS)-EDA-3 40 25 0.554 44.68Poly(NAT-GMA-BIS)-EDA-4 45 25 0.554 44.68Poly(NAT-GMA-BIS)-EDA-5 72 25 0.591 47.66Poly(NAT-GMA-BIS)-EDA-6 144 25 0.631 50.89Poly(NAT-GMA-BIS)-EDA-7 264 25 0.628 50.65

Poly(NAT-GMA-BIS)-EDA-8 2 40 0.491 39.60Poly(NAT-GMA-BIS)-EDA-9 5 40 0.488 39.35Poly(NAT-GMA-BIS)-EDA-10 17 40 0.559 45.08Poly(NAT-GMA-BIS)-EDA-11 25 40 0.632 50.97Poly(NAT-GMA-BIS)-EDA-12 48 40 1.280 100

Poly(NAT-GMA-BIS)-HMDA-1 48 40 0.919 71.80

a Diamine concentration in the reaction mixture: 0.114 g/mL of buffer.b Quantification by HCl method.c With respect to epoxy groups content on the discs determined as

1.280 mmol/g of dry polymer.

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2.4.2. Immobilization of heparin onto poly(NAT-GMA-BIS)-EDAand poly(NAT-GMA-BIS)-HMDA discs

These reactions were carried out under different reac-tion times using poly(NAT-GMA-BIS)-EDA-12 discs to yieldpoly(NAT-GMA-BIS)-EDA-12-Hep-1 and -2. Prior to thereaction, the amino-containing discs were immersed in10 mL distilled water (twice) and 10 mL 0.2 M phosphatebuffer pH 7.00 (twice) for 1 h each washes. Reductive ami-nation was then carried out by placing each poly(NAT-GMA-BIS)-EDA disc in a flask containing 5 mL of solutionof heparin (18 mg/mL) and 1.8 mg/mL NaCNBH3 preparedin the same buffer. The reactions were allowed to proceedat room temperature for 12–31 days (Table 3). Once thereactions had been completed, the discs were first washedexhaustively with 0.2 M phosphate buffer pH 7.00 until nopositive toluidine blue test was observed from the efflu-ents, and then with 0.2 M sodium acetate.

Amino groups remaining on the supports were blockedthrough acetylation. Therefore, the discs containing hepa-rin were immersed in 5 mL 0.2 M sodium acetate and the

Table 3Reaction conditions used in the immobilization of heparin on poly(NAT-GMA-BIS)-EDA and poly(NAT-GMA-BIS)-HMDA discs.

Polymer Time(days)

Hep(mg/mL)a

Hepimmobilized(lg Hep/gdry polymer)b

Poly-(NAT-GMA-BIS)-EDA-12-Hep-1

12 18 205.20

Poly-(NAT-GMA-BIS)-EDA-12-Hep-2

31 18 591.50

Poly-(NAT-GMA-BIS)-HMDA-1-Hep-3

31 18 489.90

a Heparin concentration in the reaction mixture. All experiments werecarried out using a NaBH3CN concentration of 1.8 mg/mL.

b Quantification by the toluidine blue method.

amine groups were acetylated by the addition of aceticanhydride at 5-min intervals over 25 min (five times). Dur-ing the acetylation reactions, the pH was maintained be-tween 7 and 8 by the aggregate of NaOH 5 M. Finally, thepoly(NAT-GMA-BIS)-EDA-Hep discs obtained were washedsuccessively with 0.2 M sodium acetate solution and thenwith distilled water. The supports were stored at +4 �C inethanol solution 20% vol. Once the best coupling reactionconditions for the attachment of heparin had been reached,they were used to carried out the attachment of heparin onpoly(NAT-GMA-BIS)-HMDA-1 support to yield poly(NAT-GMA-BIS)-HMDA-1-Hep-3 disc. Experimental conditionsare summarized in Table 3.

2.5. Qualitative assays for amine detection (TNBS test)

Amine-containing supports or effluents from washes ofthe amine-containing supports were analyzed in order todetect the presence of amino groups. Therefore, 0.5 mL ofsample was added to 2 mL saturated sodium borate solu-tion in a 10 mL glass test tube. Then 0.5 mL 2 mM picry-sulfonic acid in saturated sodium borate solution wasadded. The presence of amine groups on the solids or inthe effluents produced an orange color. The test was con-sidered negative when no change of color was observedwithin 10 min [41].

2.6. Quantification of amino groups on discs

The amine-group content was determined in duplicateusing the HCl-titration method for amines [42]. Therefore,0.1 g of dry sample was suspended in 5 mL HCl 0.5 M solu-tion under stirring, and the reaction occurred at reflux for1.5 h. After that, the mixture was centrifuged at1000 rpm for 15 min. Supernatant (1 mL) was then titratedusing a 0.1 M NaOH solution in the presence of phenol-phthalein indicator. The amine content of the productswas calculated from the differences found in the acidquantity of the initial and final solutions.

2.7. Quantification of heparin on discs

The amount of heparin bound to discs was determinedby a modified version of the toluidine blue method [43].The following solutions were prepared:

– 0.2% w/v NaCl solution.– Standard heparin solution: 16.6 mg of sodium heparin

dissolved in 100 mL 0.2% NaCl.– Toluidine blue solution: 12.5 mg toluidine blue dis-

solved in 250 mL 0.01 M HCl containing 0.2% NaCl.

The method was carried out at room temperature.Therefore, thirteen tubes were used in this assay. Toluidineblue solution (2.5 mL) were introduced into the tubes.Then, 60, 120, 180, 300, 350 and 400 lL of standard hepa-rin solution were added into tubes 1–6, respectively. 30 mgof dry heparin-containing supports were introduced intubes 7–9 and 30 mg of supports without heparin (control)were added into tubes 10–12. Tube 13 contained the dyesolution (used also as control). All tubes were then diluted

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with 0.2% NaCl to a total volume of 5 mL and shaken vigor-ously for 30 s. Hexane (5 mL) was added and the tubeswere shaken for further 30 s to remove the heparin-dyecomplex formed in tubes 1–6; the same procedure wascarried out with tubes 7–13 to insure the same treatment.Afterwards, tubes 7–12 were centrifuged at 1000 rpm for5 min to separate the solid polymer from the solution.The supernatant aqueous phase of all the tubes was diluted1:10 with ethanol. The absorbance was read for each sam-ple at 631 nm. With tubes 1–6, a calibration curve plottingabsorbance vs. heparin concentration and a correlationcoefficient (r) of 0.998 were obtained. Through this calibra-tion curve, the content of heparin bound to supports (tubes7–9) could be determined. Tubes 10–12 were used as con-trol to discount the toluidine blue dye not specifically ad-sorbed on the support without heparin. This polymerused as control was subjected to the same derivatizationreactions except the coupling with heparin.

2.8. Stability of heparin coupled to polymeric discs

The stability of the coupled Hep was studied. Therefore,the heparin-containing discs [poly(NAT-GMA-BIS)-EDA-12-Hep-2] were stored in two different media: ethanol20% vol and 0.2 M acetate buffer pH 5.00. The ligand con-tent was then determined at different storage times.

2.9. AT-III retention assays

Chromatography studies were performed using anÄKTA Purifier to assay the retention of AT-III. The absor-bance at 280 nm was monitored. Poly(NAT-GMA-BIS)-EDA-Hep-1, -2 (with different heparin content) and poly-(NAT-GMA-BIS)-HMDA-1-Hep-3 discs were used in theseassays. Therefore, each disc containing Hep was put intodisc housing from BIA Separations� (Ljubljana, Slovenia).Each assay was performed as follows: first, 20 mL of equil-ibrated buffer (TRIS/Citrate, pH 7.40, 0.1 M NaCl) werepumped at a flow rate of 1 mL/min through the whole sys-tem until the column was equilibrated (the absorbance at280 nm was stabilized). After that, the loading step wascarried out by injecting 5 mL of the loading solution(33 UI AT-III/mL) into the column. Then, the column waswashed with the equilibrium buffer until the absorbanceat 280 nm was zero, indicating that there was no proteinin the circulating buffer. Finally, the elution step was car-ried out with the elution buffer (TRIS/Citrate, pH 7.40,1 M NaCl), and the activity of the AT-III eluted was deter-mined with a Chromogenix-Coamatic� Antithrombin kit.A control column was also assayed. It contained a disc pre-pared under the same procedures including the aminationreaction with EDA and the coupling step with all reagents,except the addition of heparin.

3. Results and discussion

3.1. Porous properties of poly(NAT-GMA-BIS) discs

The use of macroporous polymers as supports in affinitychromatography is based on the reversible and specific

interaction between the solute to be purified and the activesites on the polymer surface. In the synthesis of porouspolymers to be used as chromatography supports in theseparation of biomolecules, it is important to obtain a spe-cial material. This should have a large pore size to allowthe diffusion of the biomolecules through the separationunit at relative low pressures, and a considerable specificsurface area where the active sites are accessible to the sol-utes in the mobile phase. However, large pore sizes do notcontribute significantly to the specific surface area. There-fore, a balance between the hydrodynamic properties ofthe support and the specific surface area should beachieved.

The porous properties of macroporous polymers can bemodified by varying the reaction parameters. For example,different porogenic mixtures are used to change the solva-tion of the polymeric chain formed during the early state ofthe polymerization reaction, and to change the porousproperties of the final product. The latter can take placesince materials with larger pore sizes are obtained with‘‘poorer” porogenic mixtures as a result of the poor sol-vency properties of the polymer chain formed [3].

In this work, macroporous polymer discs were preparedby free radical crosslinking polymerization under differentporogenic mixtures. In all cases, the monomer conversionwas close to 100%. The porous properties and polymeriza-tion conditions used are shown in Table 1.

From Table 1 it can be seen that when ethanol, pentanoland octanol were used as porogen, the products obtained[poly(NAT-GMA-BIS)-1, -2 and -3, respectively] showedvery low porosity. Similar results were found in a previouswork [29]. For these short chain alcohols, the high solubil-ity of monomers and polymers formed probably producesa phase separation (nucleation) at a later stage of the poly-merization. The polarity in the nuclei could be similar tothat of the solution; monomers could not be forced to ad-sorb preferentially into the nuclei and a larger number ofindividualized nuclei could be formed. As a consequence,a significant number of small microglobules would aggre-gate to form the final structure [44]. When dodecanoland tetradecanol were used as co-porogens, the porouspolymer obtained [poly(NAT-GMA-BIS)-4 and -5, respec-tively] showed larger porosity values (40.4% and 47.9%,respectively) and a pore size around 30–70 nm. Whenthese longer alcohol alkyl chains were used, the solubilityof the polymer formed probably decreased and polymeri-zation occurred with an early phase separation. Thus, thenuclei possibly extract the monomers from the solutioncontaining the less polar alcohol because monomers couldbe favored in the more polar medium within the nuclei. Asa result, the forming nuclei could coalesce with the chainsand therefore increase their size yielding large microglo-bules and large pores [12].

However, the recently described pore sizes yielded withdodecanol and tetradecanol were not sufficient to be usedas chromatography supports at high flow rate and lowbackpressures. Therefore, PEG 6000 was assayed as poro-gen and the macroporous polymer poly(NAT-GMA-BIS)-6so obtained presented the best porous properties of theseries. Various studies were reported in which a combina-tion of solvents with polymeric diluents yielded macropo-

Fig. 3. Scanning electron micrographs (20000 x) of poly(NAT-GMA-BIS)-4, -5 and -6 prepared at 70 �C, with dodecanol (a), tetradecanol (b) andPEG 6000 (c) as co-porogen agents, respectively.

Fig. 2. Curve of pore size distribution of macroporous poly (NAT-GMA-BIS)-6.

668 R.D. Arrua et al. / European Polymer Journal 46 (2010) 663–672

rous networks [19,29,45–47]. This is so since the role oflinear high molecular weight polymers (as an inert diluent)is to create macropores enhancing the phase separationwith the diluent. The use of polymeric porogen in the poly-merization mixture results in a phase separation at a veryearly stage of the polymerization. Additionally, the pres-ence of PEG 6000 could allow a lateral chain aggregationleading to the formation of high pores [12].

Poly(NAT-GMA-BIS)-6 has a pore size of 280.7 nm at thehighest peak in the distribution profile curve, a total spe-cific surface area of 15.4 m2/g (and 11.3 m2/g consideringthe pore sizes major than 50 nm), a pore volume of0.812 mL/g and a porosity of 51.5%. The curve of differen-tial pore size distribution can be observed in Fig. 2. Ascan be seen, the polymer presents mono-dispersed poresize distribution profile of considerable importance forchromatography applications. The content of epoxy groupswas determined using the pyridinium chloride method andwas 1.28 mmol epoxy/g of dry polymer. Fig. 3a–c show theSEM images of poly(NAT-GMA-BIS)-4, -5 and -6, respec-tively.

These polymers showed heterogeneous surfaces thatconsist of large microglobules aggregated to clusters. FromSEM images, the differences between the sizes of polymerclusters can be clearly seen, where poly(NAT-GMA-BIS)-6showed the larger sizes and consequently the greater voidsbetween them.

Since poly(NAT-GMA-BIS)-6 disc presented the bestporous properties of the series, the derivatization reactionswere continued with this product. These discs exhibiteddry masses of approximately 200 mg, 12 mm in diameterand 3 mm thick.

3.2. Derivatization reactions

The derivatization reactions carried out on the discs areshown in Fig. 4.

3.2.1. Amination reactionsAs previously reported by other studies [48], the epoxy

groups were reacted with EDA through ring opening in ba-

sic medium to introduce amine groups on the discs. In ourwork, amination reactions were previously assayed in dif-ferent buffers (phosphate 0.05 or 0.5 M pH 8.00 and car-bonate 0.05 or 0.5 M pH 9.00) and the best yield wasreached with 0.5 M phosphate buffer pH 8.00 as the med-ium. These results are not shown here [49].

To determine the influence of the diamine concentra-tion in the reaction medium, several reactions were as-sayed. In all cases an excess of diamine with respect to

Fig. 4. Derivatization reactions carried out on poly(NAT-GMA-BIS)-6 discs.

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oxirane groups in the supports was used, in order to favorone-end reaction, and therefore to reduce the crosslinkingreactions. The EDA concentration assayed varied from0.057 to 0.573 mg/mL, which represents an approximateexcess of diamine applied (with respect to oxirane groups)from 20 to 200 times, respectively. In general, an increasein the EDA concentration (by 144 h at 25 �C) yielded ahigher amount of amine groups. The results varied from0.608 (49.03% of yield) to 0.701 mmol amine/g dry poly-mer (56.53% of yield). The results do not show a significantdifference despite the very distinct concentrations of EDAused. Subsequent amination reactions were performedwith a concentration of EDA from 0.114 g/mL, representingapproximately 40-fold higher than the accessible epoxidesgroups present in poly(NAT-GMA-BIS) discs. With thisconcentration of EDA, an amine-containing disc with0.631 mmol amine/g dry polymer (50.89% of yield) wasobtained.

Then, the influence of time and temperature over theamination reaction was studied. The results are shown inTable 2. In general, longer times of reaction (using a con-centration of EDA of 0.114 g/mL, at 25 �C) yielded largeramounts of amine groups. Hence, it can be seen that whenthe attachment of EDA was at 25 �C, the larger amount ofamine groups (0.631 mmol amine/g dry support) wasyielded after 6 days of reaction. This long time of reactioncould be reduced when the reactions were carried out at40 �C, since similar coupling yields were observed after25 h of reaction (50.97% and 0.632 mmol amine/g dry sup-port). Then, at 40 �C and 48 h of reaction, the larger amine-group value (1.280 mmol amine/g dry support) was ob-tained. These experimental conditions were used for thelatter amination reactions.

As can be seen in Table 2, these experimental conditions[used to yield poly(NAT-GMA-BIS)-EDA-12] were used forthe amination reaction with the longer HMDA spacer tostudy the influence of the length of the spacer in subse-quent assays with these polymers as affinity chromatogra-phy supports. Thus, 0.919 mmol amine/g dry support,

corresponding to a yield of 71.80%, was reached. Therefore,a series of supports with a considerable amount of aminogroups and different arm lengths has been prepared.

3.2.2. Covalent immobilization of heparin onto poly(NAT-GMA-BIS)-EDA and poly(NAT-GMA-BIS)-HMDA discs

The coupling of heparin was carried out using poly-(NAT-GMA-BIS)-EDA-12 (1.280 mmol amine/g dry sup-port) and poly(NAT-GMA-BIS)-HMDA-1 (0.919 mmolamine/g dry support) discs. The attachment of heparin tothe amino-containing discs was conducted through reduc-tive amination between amine groups contained in thesupports and the aldehyde group present at the reducingend of the heparin molecule (end-point immobilization)using NaBH3CN as a reducing agent [50–54]. The amountof ligand bound to supports under different reaction timesis exhibited in Table 3.

The quantification of the amount of heparin coupled tosupports was attained by simply monitoring the toluidineblue depletion in the supernatant at 631 nm of each reac-tion and comparing it to known heparin standards [43].The procedure represents a simple assay technique whichallows the direct quantification of heparin in immobi-lized-heparin products.

It is important to note that the value calculated fromthe assay carried out on the control (polymer without hep-arin) was already discounted in the values of immobilized-heparin summarized in Table 3. The low value found in theabsence of Hep was possibly due to the little non-specificadsorption of toluidine on the polymer surface.

The greater amount of ligand coupled to poly(NAT-GMA-BIS)-EDA-12 discs was reached after 31 days of reac-tion. This long reaction time has been already reported byothers authors [55,56]. This is possibly due to the fact thatthere is no agitation during the coupling reaction and thealdehyde group of the polysaccharide is involved in theslow equilibrium with the hemiacetal form.

For poly(NAT-GMA-BIS)-HMDA-1 disc, 489.90 lg Hep/gdry polymer was reached. Therefore, the use of a longer

Table 4Stability assays on the Hep bound to macroporous poly(NAT-GMA-BIS)-EDA-12-Hep-2 discs, with an initial Hep content of 591.5 lg Hep/g drypolymer.

Medium Weeks Hep coupled(lg Hep/g drypolymer)

Hep retained (%)

Ethanol 20% vol 4 539.38 91.28 429.31 72.6

Buffer acetate0.2 M pH 5.00

4 418.77 70.88 415.27 70.2

Table 5Retention assays of AT-III with heparin-containing discs.

Supports Hepimmobilized(lg Hep/g drypolymer)

UI AT-IIIa/mLpolymer

Poly(NAT-GMA-BIS)-EDA-12-Hep-1

205.20 2.18

Poly(NAT-GMA-BIS)-EDA-12-Hep-2

591.50 3.25

Poly(NAT-GMA-BIS)-HMDA-1-Hep-3

489.90 0.55

a UI AT-III: protein activity expressed in international units, deter-mined in the elution step with buffer TRIS/citrate pH 7.40, 1 M NaCl.

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spacer did not influence the ligand coupling reaction inthese experimental conditions.

From the results mentioned, we are presenting novelmacroporous polymer discs with heparin as ligand in theirstructures. The amount of Hep coupled to poly(NAT-GMA-BIS) discs was similar to that yielded using macroporouspolymer beads [36,39,57].

In relation to the stability of the ligand coupled to poly-mers discs, Table 4 shows that the Hep content decreases9% after one month and 27% after two months in the stor-age of the support in ethanol 20% vol. On the other hand,the loss of ligand was greater using acetate buffer pH5.00, since the Hep content was reduced to 70% after fourweeks. Then, after two months of storage, the Hep contentwas kept constant.

About this, there is a previous paper [39] in which somesimilar studies of stability of heparin adsorbents were as-sayed. In that paper, the heparin content of Heparin-LiChroprep Si60 adsorbent had decreased by 32% aftertwo months when stored in 0.2 M sodium acetate at pH5. In these series of modification reactions, the blockingof free amino groups was achieved through acetylationwith acetic anhydride. The following storage in an aqueousenvironment can produce free amino groups again (on theblocked adsorbents). The leakage of the heparin ligand inthese conditions has been associated with the presenceof such free amino groups which create a hydrolytical envi-ronment for the ligand in the storage medium.

3.3. AT-III retention assays

The retention assays were performed using poly(NAT-GMA-BIS)-EDA-12-Hep-1, poly(NAT-GMA-BIS)-EDA-12-Hep-2 and poly(NAT-GMA-BIS)-HMDA-1-Hep-3 with 205.20,591.50 and 489.90 lg Hep/g dry polymer, respectively. Resultsare shown in Table 5. A column with a disc without Hep asligand, was used in the retention assays as control; it did notretain AT-III.

Comparing poly(NAT-GMA-BIS)-EDA-12-Hep-1 andpoly(NAT-GMA-BIS)-EDA-12-Hep-2 supports, it can beseen that the retention is greater for poly(NAT-GMA-BIS)-EDA-12-Hep-2 product containing a higher amount of hep-arin coupled (591.50 lg Hep/g dry polymer). This resultmay arise from the fact that the AT-III molecule would finda larger amount of interaction sites on the support. There-by, the ligand density of poly(NAT-GMA-BIS)-EDA-12-Hep-2 is roughly in three times higher than that in the case ofpoly(NAT-GMA-BIS)-EDA-12-Hep-1, whereas the AT-III

activity is higher only in 1.5 times possibly due at steric ef-fects caused by high ligand concentration limiting the in-crease of adsorbent capacity.

In order to analyze the influence of the spacer lengthused in the synthesis of supports for AT-III retention, poly(-NAT-GMA-BIS)-EDA-12-Hep-2 and poly(NAT-GMA-BIS)-HMDA-1-Hep-3 supports were compared. They present asimilar value of ligand density. A lower retention wasreached with the support-containing HMDA as spacerarm. This could be due to non-specific hydrophobic inter-actions between the hydrocarbon chain of HMDA and theprotein to be retained, which could generate conforma-tional changes in the protein structure and therefore a de-crease in the protein activity. Similar results were observedby other authors with different length spacers [17].

4. Conclusions

In this work we present the preparation of novel macro-porous poly(NAT-GMA-BIS) discs derivatized with heparinand then used in the retention of antithrombin III (AT-III).The synthesis and characterization of the macroporousbase-polymeric discs were carried out using DMSO and dif-ferent porogenic agents. The results showed that the mac-roporous polymer obtained with PEG 6000 as porogenshowed the best porous properties of the series for whichit was selected to continue the derivatization reactions.So, end-point immobilization of heparin as ligand was per-formed on the products through reductive amination.Thus, the products were reacted with two types of dia-mines: ethylenediamine (EDA) and hexamethylenedi-amine (HMDA) in order to introduce amine groups and toanalyze the spacer length influence on the immobilizationof heparin. Both the amination and the coupling of heparinreactions were performed under different conditions. Then,the higher amine-group value [1.280 and 0.919 mmolamine/g dry support for poly(NAT-GMA-BIS)-EDA-12 andpoly(NAT-GMA-BIS)-HMDA-1, respectively] was obtainedat 40 �C and 48 h of reaction. Finally, the greater amountof ligand coupled on discs was reached after 31 days ofreaction and the use of a longer spacer did not influencethe ligand coupling reaction in the experimentalconditions assayed. The amount of Hep coupled on discs[591.50 and 489.90 lg Hep/g dry polymer, for poly-(NAT-GMA-BIS)-EDA-12-Hep-1 and poly(NAT-GMA-BIS)-HMDA-1-Hep-3, respectively] was similar to that yielded

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using macroporous polymer beads. It should be noted thatalthough long periods of time were necessary, a high levelof coupling of Hep was reached. Considering the retentionof AT-III, it was greater for the product that contained ahigher amount of heparin possibly due to the largeramount of interaction sites proceeding from the ligandcoupled on the support. A lower retention was reachedwith the HMDA -containing support as spacer arm.

Therefore, these novel heparin-containing macroporouspoly(NAT-GMA-BIS) discs could be used as potential affin-ity chromatography supports. We are thus contributingwith new hydrophilic macroporous polymeric rods withpotential applications in the chromatography field.

Acknowledgements

The authors thank SECYT (Universidad Nacional de Cór-doba), FONCyT and CONICET for the financial assistance.R.D. Arrua also acknowledges receipt of a fellowship fromCONICET.

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