Materials Science and Engineering C 32 (2012) 569–573

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Materials Science and Engineering C

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Enzyme microcapsules with substrate selective permeability constructed vialayer-by-layer polyelectrolyte self-assembly

Zi-Xia Zhao a, Xin-Sheng Wang b, Xia Qin b, Qiang Chen b,⁎, Jun-ichi Anzai c

a Chinese Academy of Fishery Sciences, Beijing 100141, P. R. Chinab The Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071, P. R. Chinac Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan

⁎ Corresponding author. Tel.: +86 22 23506122; fax:E-mail address: qiangchen@nankai.edu.cn (Q. Chen)

0928-4931/$ – see front matter © 2011 Elsevier B.V. Alldoi:10.1016/j.msec.2011.12.010

a b s t r a c t

a r t i c l e i n f o

Article history:Received 30 May 2011Received in revised form 16 September 2011Accepted 8 December 2011Available online 14 December 2011

Keywords:MicrocapsulesEnzymePolyelectrolyteLayer-by-layer self-assemblyPermeability

Polyelectrolyte multilayer microcapsules with entrapped horseradish peroxidase (HRP) have been preparedvia a layer-by-layer (LbL) self-assembly strategy of polycation and polyanion on CaCO3 microparticles as tem-plates. Preparation conditions have been studied and optimized. Within the investigated ranges, use of buffersolutions with lower pH value or lower ionic strength in the buffer solution for polyelectrolyte self-assemblyhas resulted in thinner polyelectrolyte film and higher permeability of the microcapsules. For dissolving theCaCO3 templates, use of weakly acidic (pH 4.0) buffer solution in place of routinely used EDTA reagent hasimproved the catalytic activity of microcapsules. The HRP-containing microcapsules have exhibited catalyticactivity to pyrogallol as substrate, while the catalytic activity to 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sul-fonic acid) (ABTS) was severely suppressed. No less than 90% of the maximum enzymatic activity to pyrogal-lol has remained after 30 days. Promising prospects in various biocatalysis applications have been expectedfor the enzyme microcapsules, due to their selective permeability, stability and reusability.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Via a “bottom-up” fabrication strategy, various biofunctional micro-capsules of distinctive physicochemical characteristics have beendesigned by loading with proteins [1–3], nucleic acids [4–6], drugs[7–9], as well as living cells [10–12]. To meet specific demands in kindsof biotechnological applications, layer-by-layer (LbL) self-assembly hasbeen widely used as an effective technique for constructing polyelectro-lyte multilayer microcapsules with controllable structures and designa-ble functions [13–17].

A convenient method to encapsulate proteins in microcapsules wasrecently developed [18–23]. Protein-containing calcium carbonate(CaCO3) microparticles were prepared by co-precipitation of calciumchloride (CaCl2), sodium carbonate (Na2CO3), polyanion, and proteins,then the negatively charged surface of resulting CaCO3 microparticleswas coated with polyelectrolyte multilayers by an alternate adsorptionof oppositely-charged polyions. An aqueous ethylenediaminetetraaceticacid (EDTA) solution was then utilized to dissolve the CaCO3 templates,yielding hollow and porous polyelectrolyte microcapsules with proteinentrapped. Thus preparedmicrocapsules were proved of great potentialfor drug delivery and release systems [9], however, the attempt to en-capsulate enzymes for constructing microreactors and biosensors

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encountered some problems, since there was a noticeable decrease inthe detected catalysis activity after encapsulation [18,23].

In this work, horseradish peroxidase (HRP) was encapsulated as amodel enzyme, which is known to catalyze the oxidization reaction ofvarious phenolic compounds in the presence of H2O2 [24]. Conditionsfor the encapsulation of enzyme were investigated to explore their ef-fects on the catalytic performances of microcapsules. Based on thefindings, the preparation procedures of enzymatic microcapsuleswere optimized to greatly conserve the native conformation of en-zyme and provide a mild aqueous micro-environment for catalysis.Shown in Fig. 1 was the schematic illustration for the preparation ofenzymatic microcapsules, which would be a promising techniquefor encapsulation of acid-stable enzymes in industrial catalysis andenvironmental biosensing.

2. Materials and methods

2.1. Reagents

Peroxidase from horseradish (HRP, EC1.11.1.7, Type VI-A, 250–300units/mg solid for pyrogallol substrate), Albumin from bovine serum(BSA, lyophilized powder,≥96%, agarose gel electrophoresis),fluoresceinisothiocyanate-dextran (FITC-dextran, MW: 42,000), 3-mercapto-1-pro-panesulfonic acid sodium salt (MPS), 4-(2-hydroxyerhyl)piperazine-1-erhanesulfonic acid (HEPES), pyrogallol (MW: 126) and 2,2′-azino-bis(3-ethylbenz-thiazoline- 6-sulfonic acid) (ABTS, MW: 549) were pur-chased from Sigma-Aldrich Co. Poly(styrene sulfonate) sodium salt (PSS,

Fig. 1. Schematic illustration of the preparation of HRP microcapsule and its catalytic reactions.

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MW: 500,000) and Poly(allylamine hydrochloride) (PAH, MW: 10,000),Ethylene diamine tetraacetic acid sodium salt (EDTA) were purchasedfrom Scientific Polymer Products, Inc. (New York, USA), Nittobo Co.(Tokyo, Japan), and Amresco Inc. (Ohio, USA), respectively. FITC-labeledHRP (FITC-HRP) was synthesized by the coupling reaction of FITC andHRP at room temperature for 2 h in the dark [22]. All other reagentswere of analytical grade and used without further purification.

2.2. Characterization of PAH/PSS polyelectrolyte multilayers

The deposition behavior of (PAH/PSS)5 film was monitored by aquartz crystal microbalance (QCM, Model QCA917, SEIKO EG&G Co.,Tokyo, Japan). A gold film-coated 9-MHz quartz resonator was usedthroughout, in which 1 Hz of frequency change corresponds to an ad-sorption of 2.73×10−5 g/m2 of substrate.

The gold substrate of the quartz resonator was pretreated with1 mM MPS solution overnight to lead to negatively-charged surface.The LbL films were deposited on the surface of the quartz resonatorin the 1 mg/mL of PAH and PSS solutions.

2.3. Preparation of enzyme-containing microcapsules

A10 mLof 100 mMHEPES buffer solution containing 200 mMCaCl2,0.4 mg/mL HRP (or FITC-HRP) and 100 mM NaCl (pH 9.0) was quicklyadded to 10 mL distilled water containing 200 mM Na2CO3 and 2 mg/mL PSS, followed by a magnetic stirring for 30 min. The precipitatedCaCO3microparticleswere collected by centrifugation and thenwashedin 100 mM HEPES buffer solution for three times. Subsequently, (PAH/PSS)5 multilayer films were deposited on the surface of the CaCO3 mi-croparticles, by alternate adsorption of 1 mg/mL PAH (positivelycharged) in 100 mMHEPES buffer solution and 1 mg/mL PSS (negative-ly charged) in 100 mMHEPES buffer solution at different pH value andionic strength, each for 15 min. Each adsorption was followed by threetimes washing in the working buffer. The (PAH/PSS)5 film-coatedCaCO3 microparticles were collected by centrifugation and washedthree times.

The CaCO3 templates were dissolved by 100 mM EDTA solutionsvia repeated 5-min immersing and 5-min centrifugation for 3 times.An alternative dissolving step was to wash the microparticles over-night with 100 mM HEPES buffer solution (pH 4.0, NaCl 0 M), undergentle vibrating. The HRP-containing (PAH/PSS)5 microcapsulesthus prepared were collected by centrifugation and washed threetimes in 100 mM HEPES buffer solutions (pH 7.4, NaCl 0 M).

Fluorescence intensity at 520 nm of the FITC-HRP containing mi-crocapsules was determined by a fluorescence spectrophotometer(RF-5300PC, Shimadzu Co., Kyoto, Japan), at 488 nm excitation wave-length, with slit widths of 5 nm. The total quantity of encapsulated

FITC-HRP was calculated based on the fluorescence intensity of themicrocapsule suspension, according to a calibration graph which isobtained using the fluorescence intensity of free FITC-HRP. The totalweight of HRP microcapsules was measured after lyophilization.

2.4. Imaging of enzyme-containing microcapsules

FITC labeled HRP was entrapped in CaCO3 templates, which werethen coated with (PAH/PSS)5 multilayers. Before and after removal ofthe CaCO3 templates, images of microparticles and microcapsuleswere respectively taken by a microscope (Axio Imager M2, Carl ZeissInc.). Both optical and fluorescence images were obtained. The excita-tion wavelength was at 488 nm for FITC fluorescence observation.

2.5. Determination of enzymatic activity of the encapsulated HRP

The enzymatic activity of HRP-containing microcapsules for theoxidation of ABTS and pyrogallol as substrates was determined onan Ultraviolet Visible Spectrophotometer (UV-3100PC, ShimadzuCo., Kyoto, Japan).

The standard protocol p6782enz (for ABTS) and p8375enz (for py-rogallol) from Sigma was followed, when not otherwise stated. ForABTS determination, the reaction mixture (3.05 mL) contained96 mM potassium phosphate, 8.7 mM ABTS, 0.01% (w/w) hydrogenperoxide, 0.004% (w/v) BSA and 0.008% (v/v) Triton X-100. One unitof HRP will oxidize 1.0 micromole of ABTS per minute at pH 5.0 at25 °C. For pyrogallol determination, the reaction mixture (3.00 mL)contained 14 mM potassium phosphate, 0.027% (w/w) hydrogen per-oxide, 0.5% (w/v) pyrogallol and 0.04–0.07 unit peroxidase. One unitwill form 1.0 mg of purpurogallin from pyrogallol in 20 seconds at pH6.0 at 20 °C.

UV-visible adsorption spectrum of the supernatant solution wasrecorded. Enzymatic activity calculation was based on the maximumchanges of adsorption at 405 nm (ABTS dication, products of ABTS)and 420 nm (purpurogallin, products of pyrogallol) in 20 seconds,namely ΔA405 nm and ΔA420 nm.

2.6. Storage of enzyme-containing microcapsules

HRP-containing microcapsules were stored in 100 mM HEPESbuffer solution (pH 7.4, NaCl 0 M) at 4 °C. During 30 days, enzymaticactivity of the microcapsules to catalyze pyrogallol as substrate wasdetermined every other day according to the procedure describedabove. After determination, the microcapsules were washed by100 mM HEPES buffer solution (pH 7.4, NaCl 0 M) and recollectedby centrifugation.

0 1 2 3 4 5 6

0

1000

2000

3000 pH 9.0 NaCl 0.1 M pH 7.4 NaCl 0.1 M pH 7.4 NaCl 0 M

−ΔF

/ H

z

Number of (PAH/PSS) bilayers

Fig. 2. Frequency changes of gold-coated QCM resonator during the deposition of(PAH/PSS)n multilayer film in pH 7.4 and 9.0 solutions in the presence and absenceof 0.1 M NaCl (n=5).

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3. Results and discussion

3.1. QCM analysis of polyelectrolyte multilayer self-assembly

The self-assembly behaviors of (PAH/PSS)n multilayer films weremonitored by QCM using a quartz resonator at different depositionconditions. The quantity of self-assembled polyelectrolyte was re-markably influenced by the pH value and ionic strength of the poly-electrolyte solutions used for the deposition, as shown in Fig. 2.

10 µm

a

c

5 µm

Fig. 3. Optical (a, c) and fluorescence (b, d) microscope images of (PAH/PSS)5 microcapsule100 mM HEPES buffer solution (pH 4.0, NaCl 0 M).

The resonance frequency of the QCM kept a linear increase whenthe PAH/PSS film was prepared in the pH 9.0 buffer solution contain-ing 0.1 M NaCl, showing a successful deposition of the LbL multilayer.On the other hand, the amount of assembled polyelectrolyte de-creased when the film was prepared in the solution at pH 7.4 withoutNaCl. Since the ionic charges of polyelectrolyte pair (PAH/PSS) werelargely dependent on the solution pH [25], lower pH value causedweaker negative charge of polyanion and stronger positive charge ofpolycation, which led to less amount of PAH deposition and subse-quently thinner polyelectrolyte multilayer films. Ionic strength ofthe solution was also known to affect the multilayer thickness andstructure by changing the polyelectrolyte conformation [26–28].Under lower ionic strength, polyelectrolyte chains exhibited a morestretched conformation due to electrostatic repulsion, yielding thin-ner and smoother polyelectrolyte multilayer films.

3.2. Microscope images of HRP microcapsules

Fig. 3 showed optical and fluorescence microscope images of HRP-containing microcapsules, which indicated that the prepared micro-capsules were well-dispersed spheres with an average diameter ofabout 6 μm. After CaCO3 templates were dissolved by 100 mMHEPES buffer solution (pH 4.0, NaCl 0 M), the microcapsules kepttheir shape and size unchanged for at least 30 days in pH 7.4 HEPESbuffer solution. FITC-labeled HRP was used for tracking encapsulationof HRP. Fig. 3b and d showed that FITC-HRP was effectively encapsu-lated in the microcapsules.

b

10 µm

d

5 µm

s containing FITC-HRP, before (a, b) and after (c, d) CaCO3 templates were dissolved by

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Fig. 5. Catalytic activity of HRP-(PAH/PSS)5 microcapsules to pyrogallol substrates.10 μL 0.3% H2O2 solution was added into 5 mL pH 6.0 phosphate buffer containing0.2 mM pyrogallol and (1) 0.1 mL HRP microcapsule suspensions, prepared at pH 9.0,NaCl 0.1 M; (2) 0.1 mL HRP microcapsule suspensions, prepared at pH 7.4,NaCl0.1 M;(3) 0.1 mL HRP microcapsule suspensions, prepared at pH 7.4,NaCl 0 M;(4)0.1 mL buffer solutions, the reaction was taken for 5 min and the UV-visible absor-bance of supernatants was detected.

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3.3. Substrate permeability of (PAH/PSS)5 microcapsules

We have evaluated the permeability of the (PAH/PSS)5 multilayershell of HRP microcapsules using two kinds of substrates which wereknown to be oxidized by HRP. The catalytic activity of the HRPmicrocap-sules was first studied for the most commonly used substrate ABTS,which was able to be oxidized by HRP to produce green product in thepresence of H2O2. Unfortunately, however, no appreciable change incolor of the reaction mixture was detected on UV-visible absorptionspectrumwithin an hour. On the other hand, itwas found that themicro-capsules gradually turned green after several hours if the microcapsulesare not separated from the reaction mixture. Consequently, it wasstrongly suggested that the uptake of ABTS into the microcapsule wasvery slow due to a barrier of the polyelectrolyte shell. Improving perme-ability of the polyelectrolyte multilayer might improve the apparent en-zymatic activity of the microcapsules. Thus, HRP microcapsules with athinner polyelectrolyte shell were prepared using polyelectrolyte solu-tions without NaCl to use them for the oxidization reaction of ABTS, butno significant improvement in the catalytic activity was observed (Fig. 4).

Pyrogallol, another phenolic compound with lower molecularweight, was then used as substrate for HRP for the determination ofcatalytic activity. Fig. 5 shows that the HRP microcapsules exhibit sig-nificant catalytic activity to pyrogallol. Microcapsules prepared in theabsence of NaCl, which have thinner polyelectrolyte shell, exhibitedhigher catalytic activity to pyrogallol.

While ABTS and pyrogallol were both negatively charged polyphe-nolic compounds, molecular weight was the major difference be-tween the two HRP substrate, suggesting that a size exclusion mightbe the main cause of the different permeability of the two compoundsacross the polyelectrolyte shell of the microcapsules. It is very slowfor molecules with larger sizes to get access to the enzyme entrappedinside the microcapsules, such as ABTS. As a result, little catalytic ac-tivity was able to be detected. Similar selective permeability of poly-electrolyte multilayer films was reported for LbL film-modifiedelectrodes and for polyelectrolyte microcapsules [29–31]. The selec-tive permeability of the polyelectrolyte shell suggested a potential ap-plication of enzyme microcapsules for the development of substrate-specific biocatalysts.

3.4. Improvement of catalytic activity for HRP-containing microcapsules

The CaCO3 templates were dissolved often by using EDTA solutionfor producing hollowmicrocapsules [20–25], because EDTA is a chelat-ing agent of metal ions such as Ca2+, Mg2+, Fe2+ and Fe3+. EDTA was

200 300 400 500 600 700 800

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Fig. 4. Catalytic activity of HRP-(PAH/PSS)5 microcapsules to ABTS substrate. 10 μL 0.3%H2O2 solution was added into 5 mL pH 5.0 phosphate buffer containing 0.2 mM ABTSand (1) 0.1 mL HRP microcapsule suspensions, prepared at pH 9.0,NaCl 0.1 M;(2)0.1 mL HRP microcapsule suspensions, prepared at pH 7.4,NaCl 0.1 M;(3) 0.1 mL L HRPmicrocapsule suspensions, prepared at pH7.4,NaCl 0 M;(4)0.1 mL buffer solutions, the re-action was taken for 5 min and the UV-visible absorbance of supernatants was detected.

also known to be an inhibitor formetal ion-dependent enzymes [32,33].For preparing HRP microcapsules, we tried to dissolve the CaCO3 tem-plate with a slightly acidic HEPES buffer solution (100 mM HEPES, pH4.0) as an alternative for EDTA solution. The CaCO3 template wasfound to be slowly dissolved in the HEPES buffer solution. No lessthan 8 h processing in the HEPES buffer solution under gentle vibratingwas essentially required for dissolving the CaCO3 template thoroughly.Fig. 6 showed the catalytic activity to pyrogallol of the HRP microcap-sules processed in 0.1 M EDTA and in pH 4.0 HEPES solutions. Catalyticactivity of the HRPmicrocapsule processed in the HEPES buffer was ap-parently higher than that of EDTA-processed microcapsule. These re-sults showed that the pH 4.0 HEPES buffer was effective to prepareHRP microcapsules with improved catalytic activity although the pro-cessing was somewhat time-consuming. EDTA might have undesirableeffect on the catalytic activity of HRP in the microcapsules.

In contrast, no apparent improvement was observed in the cata-lytic activity of the HRP microcapsules to ABTS even if the HEPES buff-er was employed for dissolving CaCO3 (data not shown). This verifiedagain that the apparent catalytic activity of HRP microcapsules toABTS was mainly limited by the low permeability of ABTS across thepolyelectrolyte shell.

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Fig. 6. Catalytic activity of HRP-(PAH/PSS)5 microcapsules to pyrogallol substrates.10 μL 0.3% H2O2 solution was added into 5 mL pH 6.0 phosphate buffer containing0.2 mM pyrogallol and (1) 0.1 mL HRP microcapsule suspensions, processed by 0.1 MEDTA solution; (2) 0.1 mL HRP microcapsule suspensions, processed by 100 mMHEPES buffer solution (pH 4.0, NaCl 0 M);(3) 0.1 mL buffer solutions, the reactionwas taken for 5 min and the UV-visible absorbance of supernatants was detected.

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3.5. Storage of HRP-containing microcapsules

The reusability and stability of the HRP microcapsules stored in100 mM HEPES buffer solution (pH 7.4) at 4 °C was tested (n=5),using pyrogallol as substrate. The total amount of encapsulated HRP in1 mL of microcapsule suspension was calculated to be 0.21 mg, accord-ing to thefluorescence intensity. Enzymatic activity of the same amount(0.21 mg) of fresh and free HRP to pyrogallol was determined to be 12.1units, while the HRP microcapsules exhibited 9.51 units of enzymaticactivity on the first day.

Enzymatic activity of HRP microcapsules was determined everyother day. After determination, microcapsules were recollected by asimple centrifugation and washed in 100 mM HEPES buffer solution(pH 7.4, NaCl 0 M). After continuous determination in 30 days (reusedfor 15 times), 91% of the maximum activity was detected for the HRPmicrocapsule, indicating that the bioactivity of HRP can be successfullyconserved in the microcapsules. Since free HRP could not be reused, westored free HRP in the same buffer solution (pH 7.4, 100 mM HEPES,NaCl 0 M) at 4 °C for 30 days, while the enzymatic activity remained68% of its maximum activity.

Thus, the HRPmicrocapsules exhibited significant advantages in therepeated usage after recovery by a simple centrifugation and washing.

4. Conclusions

An improvedmethod for the preparation of HRP microcapsules wasestablished in this work based on the LbL polyelectrolyte self-assembly.The polyelectrolytemultilayer shell of themicrocapsule exhibited selec-tive permeability to ABTS and pyrogallol probably due to size exclusion,resulting in an apparent specificity in the catalytic reaction of HRP. An-other finding is that the catalytic activity of the microcapsule was en-hanced when the CaCO3 template was dissolved in weakly-acidicHEPES buffer solutions in place of EDTA solutions. The excellent reus-ability and stability of the HRP microcapsules would contribute totheir potential use in the monitoring and treatment of phenolic pollut-ants in water [23]. And the proposed method would be useful for effec-tive encapsulation of various acid-stable enzymes.

Acknowledgements

The financial supports by National Natural Science Foundation ofChina (No: 30870665), Natural Science Foundation of Tianjin (No:09ZCGHHZ00100), Special Scientific Research Funds for Central Non-profit Institutes, Chinese Academy of Fishery Sciences (No: 2010C019)are well acknowledged.

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