23

5

Research ArticleReceived: 27 January 2011 Revised: 4 May 2011 Accepted: 7 July 2011 Published online in Wiley Online Library: 3 October 2011

(wileyonlinelibrary.com) DOI 10.1002/pi.3177

Encapsulation of urease in double-layeredhydrogels of macroporous poly(2-hydroxy-ethyl methacrylate) core and poly(ethyleneoxide) outer layer: fabrication and biosensingpropertiesPetar Petrov,∗ Denitza Jeleva and Christo B Tsvetanov

Abstract

Double-layered hydrogels of super-macroporous poly(2-hydroxyethyl methacrylate) (PHEMA) cryogel core and poly(ethyleneoxide) (PEO) hydrogel outer layer for encapsulation of the enzyme urease were constructed. The enzyme was entrapped intothe pores of PHEMA cryogel by soaking and then the core was covered with a PEO layer. The leaking of urease from the core wasprevented when the density of the PEO network was increased by incorporation of the crosslinking agent, poly(ethylene glycol)diacrylate. The hybrid system exhibited a nearly constant enzyme activity with time and maintained its structural integrity afterseveral reactions of hydrolysis of urea. The potential of double-layered hydrogels containing urease for establishment of waterpollution with copper was investigated as well.c© 2011 Society of Chemical Industry

Keywords: hydrogels; cryogels; enzymes; urease; biosensors

INTRODUCTIONHydrogels are polymeric networks that absorb water whileremaining insoluble and preserving their characteristic three-dimensional structure because of the large number of physicalor chemical links between the polymer chains.1 Hydrophilicityis one of the main factors that determines the biocompati-bility of hydrogels and makes them attractive for applicationin medicine and pharmacy as drug carriers, tissue engineeringmatrices, membranes for biosensors, contact lenses, wound dress-ings, cell carriers etc.1 – 4 A new class of hydrogels, the so-calledsuper-macroporous hydrogels, has received considerable inter-est in the last two decades due to their unique heterogeneousopen porous structure which significantly increases equilibriumsorption properties and allows unhindered diffusion of solutes,nanoparticles and even microparticles.5 Such materials have beenexploited for many applications, especially in biomedicine andbiotechnology for immobilization of biomolecules, drugs, cellsetc.6

In general, the immobilization of biomolecules in a supportmatrix is among the preferred techniques employed to im-prove their stability and performance under both storage andoperational conditions. For instance, immobilized biocatalystshave some significant advantages over free biocatalysts likeeasy separation from the reaction medium and the productobtained, possibility of reuse for many reaction cycles, bet-ter control by means of activity, specificity, stability etc.7 Onthe other hand, a number of synthetic hydrogels possess highchemical, mechanical and biological stability, good biocompat-ibility and do not interfere with the native structure of the

enzyme protein. Therefore, many of the developed hybrid sys-tems containing biomolecules are on the basis of synthetichydrogels.8

The immobilization of biocatalysts in polymer hydrogels hasbeen carried out by entrapment, physical adsorption, encapsu-lation or covalent attachment.9 Immobilization of enzymes viaformation of covalent bonds prevents the release of enzyme intothe solution upon use due to the stable nature of the bondsformed between biomolecules and matrix. However, in order toachieve high levels of activity, the amino acid residues essentialfor catalytic activity must not be involved in the covalent linkageto the support. In addition, due to the structural changes inducedby the interaction of the macromolecules with the support, a fallin activity has been observed.10,11 The entrapment of enzymesinto polymer hydrogels is based on the occlusion of an enzymewithin a polymeric network that allows the substrate and prod-ucts to pass through, but retains the enzyme. In this case, theperformance of the system depends strongly on the enzyme re-tention, its specific activity, and the diffusibility of the substratesand products. In some cases an inhibition of catalytic activity hasbeen registered upon reuse of material. Usually, the encapsulationmethod consists in free enzyme molecules located in a confined

∗ Correspondence to: Petar Petrov, Institute of Polymers, Bulgarian Academy ofSciences, ‘Akad. G. Bonchev’ Str. 103A, 1113 Sofia, Bulgaria.E-mail: ppetrov@polymer.bas.bg

Institute of Polymers, Bulgarian Academy of Sciences, ‘Akad. G. Bonchev’ Str.103A, 1113 Sofia, Bulgaria

Polym Int 2012; 61: 235–239 www.soci.org c© 2011 Society of Chemical Industry

23

6

www.soci.org P Petrov, D Jeleva, CB Tsvetanov

space limited by a semi-permeable membrane. The membraneallows diffusion of small molecules of the substrate and productsfrom the reaction inside the capsule and vice versa. One majorproblem of this method is that with increasing concentration ofsome of the final products in the capsule some rupture of themembrane can occur.

The present work reports on a method for encapsulationof a relatively large amount of enzyme into hybrid double-layered hydrogels. Urease, a model nickel-dependent enzymewhich catalyzes the hydrolysis of urea to ammonia and carbondioxide,12 was entrapped into the pores of super-macroporouspoly(2-hydroxyethyl methacrylate) (PHEMA) cryogel and thencovered with a thin semi-permeable layer of poly(ethyleneoxide) (PEO) hydrogel. PHEMA cryogels were obtained by UVirradiation of moderately frozen systems, using hydrogen peroxideas a source of radicals, and subsequent thawing,13 while PEOhydrogels were synthesized by UV irradiation of PEO aqueoussolution in the presence of an aromatic photo-initiator – (4-benzoylbenzyl)trimethylammonium chloride (BBTMAC).14

EXPERIMENTALMaterials2-Hydroxyethyl methacrylate (HEMA), poly(ethylene glycol) di-acrylate (average molecular weight 575 g mol−1) and (4-benzoylbenzyl)trimethylammonium chloride were purchasedfrom Aldrich (Sofia, Bulgaria) and used without purification.Poly(ethylene oxide) (molecular weight 1×106 g mol−1) was kindlydonated by Union Carbide Corporation. Urease (EC 3.5.1.5 fromJack Bean), CuSO4.5H2O and H2O2 (30 vol% water solution) werereceived from Merck.

Synthesis of PHEMA cryogel1.5 g HEMA was dissolved in 15 mL deionized water at roomtemperature. Then, 0.15 g crosslinking agent (poly(ethylene glycol)diacrylate, PEGDA) and 0.3 mL initiator (30 vol% H2O2) were addedunder stirring to obtain a homogeneous aqueous solution. Then1 mL portions of the solution were poured into Teflon dishes(12 dishes with diameter of 20 mm) forming a thick layer, andwere frozen at −20 ◦C for 2 h. The dishes were irradiated with fullspectrum UV-visible light with Dymax 500-EC UV curing equipmentfor 5 min (irradiation dose 28.5 J cm−2). The samples were thenimmersed in deionized water for 3 days at room temperature andthe water was replaced every 24 h to wash out the unreactedchemicals. Finally, PHEMA cryogels were freeze dried in an Alpha1–2 Freeze Drier (Martin Christ) at −55 ◦C and 0.02 mbar (2Pa) for24 h.

Preparation of hybrid double-layered hydrogels containingureaseLyophilized PHEMA cryogel was immersed in 3 mL freshly preparedsolution of enzyme in distilled water (1.5 mg mL−1) for 30 min withcontinuous shaking. Then the discs were weighed to determinethe amount of enzyme taken and frozen for 2 h at −20 ◦C.

Separately, a 3 wt% solution of PEO was prepared as follows:0.3 g of polymer was added to 8 mL of distilled water whilestirring at high speed for 1 min. The resulting mixture was kept for24 h at 4 ◦C to dissolve the polymer and obtain a homogeneoussolution. Then, 0.015 g (4-benzoylbenzyl)trimethylammoniumchloride (dissolved in 2 mL distilled water) with a given amount ofPEGDA (0, 75 and 100 wt% of PEO) was added.

Each frozen disc of PHEMA cryogel containing urease was placedin a Teflon dish and evenly coated on all sides with 1.8 mL PEOsolution, and the samples were subsequently irradiated with fullspectrum UV-visible light for 4 min on one side and 2 min on thesecond side.

For the detailed characterization of PEO hydrogel (gel fractionyield and degree of swelling), the gels were synthesized separately(without core) following the same experimental conditions.

Measurements of gel fraction and degree of swellingThe gel fraction (GF) yield and degree of swelling (DS) of PEOhydrogels were determined gravimetrically. The GF was estimatedby weighing the insoluble part after extraction for a few days atroom temperature.

GF yield (%) = weight of dried sample

initial weight of polymer(+ crosslinker)× 100

The degree of swelling was determined as follows. Discs ofdried gels were weighed and then immersed in distilled water atroom temperature and an equilibrium water uptake was reached.The surface of the cryogel was blotted with filter paper prior toweighing.

DS = weight of swollen sample

weight of dried sample

Surface morphologyPHEMA cryogels were frozen in liquid nitrogen, fractured andfreeze dried in an Alpha 1–2 Freeze Drier (Martin Christ) at −55 ◦Cand 0.02 mbar (2Pa) for 24 h. Then the specimens were fixedon a glass substrate and coated with gold for 60 s. The interiormorphology of the gels was studied using a JEOL JSM-6390scanning electron microscope operating at 5 kV.

Protein assayThe amount of urease released by the hybrid hydrogels wasdetermined by the biuret method. The method is based onspectrophotometric measurement at 330 nm of the blue-purplecolour resulting from the complex formed between the peptidebonds of protein and copper ions from the Benedict reactant, inalkaline medium.15

Activity assay of free and immobilized ureaseThe activities of free and immobilized urease were determinedusing Nessler’s method.11 The method is based on the ammoniumliberated from urea hydrolysis, determined spectrophotometri-cally at a wavelength of 405 nm by measuring the intensity of theyellow-coloured compound formed after the addition of Nessler’sreagent.

Free urease activity0.1 mL of enzyme solution (1.5 g L−1) was added to 0.9 mL ofphosphate buffer (50 mM, pH 7.4) and incubated with 1 mL ofurea solution (3.2 g L−1) for a given temperature and time. Theenzymatic reaction was terminated by addition of 1 mL 10%trichloroacetic acid. An aliquot (1 mL) of the reaction mixture wastransferred to a 50 mL volumetric flask, 1 mL Nessler’s reagent wasadded and then the volume was made up to 50 mL with distilledwater.

wileyonlinelibrary.com/journal/pi c© 2011 Society of Chemical Industry Polym Int 2012; 61: 235–239

23

7

Encapsulation of urease in double-layered hydrogels www.soci.org

Figure 1. Digital image of a hybrid hydrogel of a PHEMA core and PEOouter layer.

Immobilized urease assayFor the determination of immobilized urease activity discs ofhybrid hydrogels containing urease were incubated in 3 mL ureasolution (3.2 g L−1) at room temperature for 24 h. After that, thegels with immobilized enzyme were separated from the reactionmixture and kept in a freezer. An aliquot of the reaction mixturewas assayed analogically to the free urease.

EA(µmol min−1) = �A.V.MA

t.v.l× 1000

where EA is enzyme activity, �A is the absorbance change, V is thetotal reaction volume, MA is the molar absorptivity, v the volumeof sample used, t the incubation time and l the length of thecuvette.

Determination of the degree of inhibition of EA by copper16

Hybrid hydrogels containing urease were incubated in an aqueoussolution of CuSO4.5H2O (Cu2+ concentration 0.01–1 mg L−1) for30 min. The degree of inhibition was determined from

Einh(%) = EA1 − EA2

EA1× 100

where EA1 is the enzyme activity of the non-inhibited sample andEA2 is the enzyme activity after the inhibition test.

RESULTS AND DISCUSSIONSystems based on physically entrapped enzyme molecules in aswollen polymer network have to overcome several crucial issuesrelating to leaking of enzyme from the carrier, unhindered diffusionof the substrate and reaction products to/from the enzyme, long-term enzyme activity etc.7 PHEMA and PEO are biocompatiblehydrophilic polymers widely used in biotechnology and medicine,2

and in this aspect are suitable for fabrication of a polymer matrixfor encapsulation of biomolecules. In this study, double-layeredsystems consisting of a super-macroporous PHEMA cryogel corecontaining urease and an outer layer of PEO hydrogel (Fig. 1) wereprepared employing the UV irradiation technique14 and tested formanifold hydrolysis reactions of urea in a batch reactor.

First, PHEMA cryogel was synthesized by irradiation of a frozenaqueous solution of monomer, H2O2 and crosslinking agent

Figure 2. SEM micrograph of PHEMA cryogel (initial monomer concentra-tion 10 wt%, temperature of freezing −20 ◦C, H2O2 5 wt% and PEGDA 10wt% to the monomer).

with UV light. The polymer network is formed by a free radicalpolymerization/crosslinking reaction initiated by the UV light. Assynthesized, PHEMA cryogel is a macroporous opalescent materialconsisting of dense polymer walls surrounding interconnectedpores (Fig. 2). We assume that the structure of the freezedried PHEMA cryogels used for constructing hybrid hydrogelsclosely resembles the morphology visualized by SEM since bothspecimens were obtained by the freeze drying technique. Thus,PHEMA cryogel, due to its interconnected pores, provides anopportunity to accommodate a large amount of enzyme solution(more than 90% of the gel weight). In addition, in water PHEMAcryogels maintain their initial volume obtained according to thesynthesis procedure (no additional swelling is observed),13 whichfacilitated the formation of an outer layer. The pores of lyophilizedPHEMA cryogel discs were filled with ca 0.5 mL solution of enzymeby soaking in a freshly prepared aqueous solution of urease(1.5 mg mL−1), as schematically illustrated in Fig. 3, followed byfreezing at −20 ◦C. Then, the frozen cryogel discs containingurease were placed in a concentrated PEO solution to form aneven coating on each side. Finally, the PEO hydrogel was obtainedvia UV irradiation. The high viscosity of PEO solution was thedetermining factor to avoid penetration of PEO solution into thecore during the synthesis procedure. The PEO hydrogel obtainedhas to function as a semi-permeable membrane that preventsleaking of urease from the PHEMA core and, on the other hand,allows diffusion of the substrate and reaction products throughthe outer layer.

The efficiency of urease retention by the PEO layer wasquantified by the incubation of hybrid hydrogels, containingurease, in water for 24 h and a subsequent determination ofthe protein content in the extract. Thus, one may estimate theamount of urease, if any, released by the polymer carrier. Ourinitial experiments revealed certain leaking of urease (ca 20%)within the time interval studied (Table 1), which indicates that thepolymer network is not sufficiently dense to restrict the diffusionof enzyme molecules. Therefore, in order to increase the densityof the PEO network, a crosslinking agent (PEGDA) was addedto the initial PEO solution. Expectedly, the bifunctional PEGDAwas incorporated into the PEO network as indicated by GF yieldvalues approaching 99% and the twofold decrease of DS (Table 1).At a certain content of PEGDA the mesh size of the polymer

Polym Int 2012; 61: 235–239 c© 2011 Society of Chemical Industry wileyonlinelibrary.com/journal/pi

23

8

www.soci.org P Petrov, D Jeleva, CB Tsvetanov

Figure 3. Sketch of entrapment of urease in PHEMA cryogel pores: (a) urease solution; (b) soaking of freeze dried cryogel in urease solution; (c) cryogelcontaining urease in the pores.

Table 1. Synthesis and properties of PEO hydrogel membrane via UVirradiation at room temperature

PEO : PEGDA weightratio

GF yield(%)

Degree ofswelling

Relative retention(%)

1 : 0 94 ± 2 71 ± 6 80 ± 3

1 : 0.75 98 ± 2 40 ± 4 83 ± 3

1 : 1 99 ± 2 37 ± 4 100 ± 2

Initial PEO concentration 3 wt%, irradiation time 6 min; BBTMAC 5 wt%to polymer.

network becomes as low as the hydrodynamic diameter of thebiomolecules and the latter cannot pass through the hydrogel.It was established that the urease molecules are quantitativelyretained in the PHEMA core at a PEO : PEGDA weight ratio of 1 : 1(Table 1).

In a control experiment we attempted to construct hybrid gelswithout PHEMA, i.e. to cover only frozen enzyme solution (0.5 mL)with PEO/PEGDA outer hydrogel. In this case, during irradiationof the samples with UV light the frozen enzyme solution (core)melted quickly and mixed with the PEO solution before onsetof the crosslinking reaction. Thus, the resulting material had animpaired integrity of structure and released the whole contentof enzyme. This experiment demonstrates the crucial role of themacroporous PHEMA scaffold necessary for the construction ofhigh quality hybrid gels containing urease.

The changes in enzyme activity with time are among the mostimportant technological characteristics concerning the processesof urea hydrolysis. We found that EA of free urease decreasedby a factor of 3 upon storage of enzyme solution in a refrigerator(+4 ◦C) for 7 days. Figure 4 shows EA of urease freshly encapsulatedin hybrid double-layered hydrogel and the activity of the samesample after 7 days’ storage (+4 ◦C). Obviously the enzymemaintains a constant EA in the time interval studied which suggeststhat the hybrid hydrogels provide an improved stability of ureasein terms of long-time storage and reuse for hydrolysis of urea.

Another test demonstrating the benefit of encapsulation is thestudy of EA in several continuous hydrolysis reactions (cycles)(Fig. 5). The test involves consecutive incubation of hybrid gels in10 mL urea solution (3.2 g L−1) for three cycles of 24 h where afterevery cycle the samples were placed immediately in fresh reactionmedium. A slight gradual decrease of EA was observed, resultingin 35% reduced activity after the third cycle. Such behaviourmight be attributed to a progressive accumulation of reactionproducts in the hydrogel that leads to inhibition and decreased

Figure 4. Comparison of the urease activity of the hybrid double-layeredsystem, before and after storage for 7 days, incubated in 10 mL ureasolution (3.2 g L−1).

Figure 5. Effect of number of cycles on the enzymatic activity of the hybriddouble-layered system incubated in 10 mL urea solution (3.2 g L−1).

reaction rate, respectively. Indeed, on incubation of gels in distilledwater to wash the reaction products, urease regained its initialvalue of EA. We should mention that during the experimentsthe hybrid hydrogels maintained integrity and no rupture of theouter PEO layer was detected. These results undoubtedly show

wileyonlinelibrary.com/journal/pi c© 2011 Society of Chemical Industry Polym Int 2012; 61: 235–239

23

9

Encapsulation of urease in double-layered hydrogels www.soci.org

the potential of hybrid hydrogels containing urease for manifolduse in processes of decomposition of urea.

The hybrid system developed can be exploited as biosensors16,17

for establishment of water pollution with heavy metals. It isknown that heavy metals have an inhibitory effect on urease,where the strongest inhibitors are Hg, Ag and Cu.18 – 20 Usually,the heavy metal ions block the thiol groups in the proteins. Inaddition, some ions like Cu2+ and Zn2+ have high affinity to N-containing ligands16 and form strong complexes that also causeinhibition of urease. It has been reported that the concentrationof heavy metals giving 50% inhibition (IC50) of free urease is 2orders of magnitude lower than urease covalently immobilizedon inorganic or organic carriers.18,19 The difference is attributedto structural changes introduced to the enzyme by the appliedimmobilization procedure resulting in lower accessibility of activesites. The physically entrapped enzyme molecules are much lessrestricted than the covalently bound ones and one may expectthat the urease encapsulated in hybrid hydrogels would haveIC50 values comparable with that of free urease. We assessed thesensitivity of our model system (disc with diameter 25 mm andthickness 8 mm; see Fig. 1) to detect Cu2+ in the concentrationrange 0.01–1 mg L−1. It was found that IC50 is approximately0.02 mg L−1 and that value is of the same order as for free urease.19

It seems that the hybrid system developed is sensitive enough todetect a very low contamination of heavy metal ions and has goodpotential for application as a biosensor.

CONCLUSIONSHybrid double-layered hydrogel consisting of a super-macroporous PHEMA cryogel core containing urease and an outerlayer of PEO hydrogel was fabricated via the UV irradiation tech-nique. The macroporous PHEMA cryogel scaffold accommodateda large amount of enzyme in its pores. To restrict the leaking ofurease from the core, the mesh size of the PEO layer was de-creased by addition of the crosslinking agent PEGDA. The hybridhydrogel system exhibited a constant enzyme activity with timeand potential for manifold reuse in processes of decomposition ofurea.

The potential of double-layered hydrogel containing urease forestablishment of water pollution with heavy metals such as copperwas confirmed. The model system is sensitive enough to detectcontamination of Cu2+ at contents as low as the content detectedby free urease.

ACKNOWLEDGEMENTSThe financial support of the National Science Fund of Bulgaria(BY-X-302/07) is gratefully acknowledged. P.P. thanks ProfessorR. Dimkov and his co-workers for their assistance and fruitfuldiscussions.

REFERENCES1 Zavan B, Cortivo R and Abatangelo G, Hydrogels: Biological Properties

and Application, ed. by Barbucci R. Hydrogels and Tissue Engineering,Springer-Verlag Italia, Milan, pp. 1–9 (2009).

2 Hoffman AS, Adv Drug Delivery Rev 43:3–12 (2002).3 Peppas NA, Bures P, Leobandung W and Ichikawa H, Eur J Pharm

Biopharm 50:27–46 (2000).4 Peppas NA, Hilt JZ, Khademhosseini A and Langer R, Adv Mater

18:1345–1360 (2006).5 Lozinsky VI, Russ Chem Rev 71:489–511 (2002).6 Lozinsky VI, Galaev IY, Plieva FM, Savina IN, Jungvid H and

Mattiasson B, Trends Biotechnol 21:445–51 (2003).7 Mateo C, Palomo JM, Fernandez-Lorente G, Guisan JM and Fernandez-

Lafuente R, Enzyme Microb Technol 40:1451–63 (2007).8 Sheldon RA, Adv Synth Catal 349:1289–307 (2007).9 Brena BM and Batista-Viera F, Immobilization of Enzymes and Cells, 2nd

edition, ed. by Guisan JM. Immobilization of Enzymes: A LiteratureSurvey, Humana Press, Totowa, NJ, pp. 15–30 (2006).

10 Dumitriu S, Popa M, Artenie V and Dan F, Biotechnol Bioeng 34:283–90(1989).

11 Tyagi C, Tomar LK and Singh H, J Appl Polym Sci 111:1381–90 (2009).12 Krajewska B, J Mol Catal B: Enzym 59:9–21 (2009).13 Petrov P, Petrova E and Tsvetanov CB, Polymer 50:1118–23 (2009).14 Doycheva M, Petrova E, Stamenova R, Tsvetanov CB and Riess G,

Macromol Mater Eng 289:676–80 (2004).15 Herbert D, Phipps PJ and Strange RE, Methods in Microbiology, ed.by

Norris JR and Ribbons DW. Chemical analysis of microbial cells,Academic Press, London/New York, pp. 209–344 (1971).

16 Park B-W, Yoon D-Y and Kim D-S, Biosens Bioelectron 26:1–10 (2010).17 Kato M, Shoda N, Yamamoto T, Shiratori R and Toyooka T, Analyst

134:577–81 (2009).18 Gabrovska K and Godjevargova T, J Mol Catal B: Enzym 60:69–75

(2009).19 Jung K, Bitton G and Koopman B, Water Res 29:1929–33 (1995).20 Fahmy AS, Bagos VB and Mohammed TM, Bioresour Technol 64:121–9

(1998).

Polym Int 2012; 61: 235–239 c© 2011 Society of Chemical Industry wileyonlinelibrary.com/journal/pi