Fabrication of Polysaccharide-inorganic Hybrid Biocapsules with

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Fabrication of Polysaccharide-inorganic Hybrid Biocapsules with Improved Catalytic Activity and Stability Yanjun Jiang, Lei Zhang, Dong Yang, Lin Li, Yufei Zhang, Jian Li, and Zhongyi Jiang* Key Laboratory for Green Chemical Technology of Ministry of Education, School of Chemical Engineering and Technology, Tianjin UniVersity, Tianjin 300072, P. R. China

In this study, a novel kind of calcium phosphate-mineralized chitosan/alginate microscapsules was prepared through a bio-inspired process for efficient encapsulation of yeast alcohol dehydrogenase (YADH). In this biomimetic process, when Ca2+-containing chitosan droplets were added into phosphate-containing solution of alginate, a thin chitosan/alginate film formed immediately around the microcapsules coupled with in situ precipitation of calcium phosphate. The biomineralization of calcium phosphate was mimicked by the counterdiffusion system in which Ca2+ ions and phosphate ions migrated into the chitosan/alginate film from opposite directions, respectively. Formation of a microcapsule was confirmed using a Zoom Stereo Microscope, the surface morphology of the microcapsule was characterized by SEM, the surface element composition of microcapsules was analyzed by XPS, and the pore-size distribution of the microcapsule shell was determined by BET. YADH encapsulated in the microcapsules exhibited significantly higher activity and recycling stability in a broader temperature range than the free YADH. We believe that such hybrid materials will find promising applications in enzyme encapsulation and drug delivery systems. 1. Introduction The polymer-inorganic hybrid carrier has found increased application in enzyme immobilization due to its moderate hydrophilicity, controllable transport characteristics, and good physicochemical stability,1-3 which could create a suitable environment for the enzyme.4-6 At present, two kinds of configurations can be found for polymer-inorganic hybrid carriers: the mixed-matrix configuration7,8 and the core-shell configuration.9-11 Compared with the mixed-matrix configuration, the core-shell configuration can create a more naturelike environment for the immobilized enzyme. So far, the sol-gel method constitutes the most popular technique for preparation of the inorganic shell.12-14 However, the necessity of using a strong acid or base as the catalyst in the conventional sol-gel method seriously hinders its wide application in enzyme encapsulation. Fortunately, biomineralization15,16 in nature has offered a delicate prototype of the inorganic mineral formation in organisms. As we all know, the most important aspects in biomineralization are the controlled nucleation and growth of inorganic minerals from aqueous solutions under the mediation of an organic matrix secreted by the cell and the formation of the biomineralized materials with the hierarchical structure and specific assembly. Consequently, biomimetic synthesis inspired by the biomineralization has now become a promising field in inorganic materials chemistry research. Vast reports on mineralized biopolymer capsules as encapsulation carrier can be found spanning from material design,17-19 structure manipulation,10,20 to process intensification.21,22 Chitosan and alginate are natural cationic or anionic polysaccharides, respectively, which have been successfully utilized in enzyme immobilization23-25 and other biomaterials26-28 attributed to their superior biocompatibility. Furthermore, alginate has been regarded as an efficient structure-directing agent for inorganic minerals.29 For example, alginate gels have already been used to control the growth of monometallic and bimetallic * To whom correspondence should be addressed. Tel.: 86-22-2789 2143; Fax: 86-22-2789 2143. E-mail: [email protected].

nanoparticles such as gold, silver, cobalt, and nickel.30 However, as far as we are concerned, alginate has not yet been utilized as the template for the amorphous calcium phosphate deposition. Calcium phosphate, the main inorganic component of bone, is of superior biocompatibility, insolubility, and mechanical stability, which has been proven to be suitable for the enzyme immobilization.31 Compared with silica, the common material in immobilized enzyme, calcium phosphate is of good biocompatible and can be prepared in mild conditions. In this study, polysaccharide-inorganic hybrid biocapsules were fabricated by a one-pot method.16,20,21 The formation of the alginate/chitosan film due to the electrostatic interaction can be coupled with the controlled precipitation of calcium phosphate arising from the counter-diffusion of ions across the polysaccharide interface. Nucleation of calcium phosphate occurring within the film produced mineralized microcapsules. The hydrophilicity and biocompatibility of the chitosan core ensured a comfortable microenvironment for YADH,32 whereas the alginate-templated calcium phosphate outer shell created a cage for the enzyme. To investigate the catalytic performance and the relevant stability of enzyme-contained hybrid microcapsules, the reversible conversion of formaldehyde into methanol using YADH (EC 1.1.1.1, from Saccharomyces cereVisiae) as a catalyst and nicotinamide adenine dinucleotide (NADH) as a coenzyme was employed. 2. Experimental Section 2.1. Materials. Yeast alcohol dehydrogenase (YADH, EC1.1.1.1), nicotinamide adenine dinucleotide (NADH, grade I, 98%), and chitosan (deacetylation degree 75-85%, viscosity 20-200 cps) were purchased from Sigma-Aldrich. Sodium alginate (average molecular weight, 6.27 × 105) was purchased from Shanghai Tianlian, China. Other reagents were of analytical grade and used without further purification. 2.2 Preparation of hybrid microcapsule. Sodium alginate was dissolved in deionized water at a final concentration of 0.8% (w/v) unless otherwise noted. Calcium phosphate-mineralized chitosan/alginate microcapsules were prepared by adding drop-

10.1021/ie071315r CCC: $40.75 © 2008 American Chemical Society Published on Web 03/18/2008

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Figure 1. Schematic representation for the formation process of calcium phosphate-mineralized chitosan/alginate microcapsule.

lets of chitosan (2%(w/v)) containing CaCl2 (final concentration was between 0.05 and 0.2 M) into an aqueous solution of sodium alginate containing Na2HPO4 (final concentration was 60 mM), using a 10 mL syringe through a 0.9 mm diameter needle under constant stirring at room temperature. The droplets were left for 10 min in the sodium alginate solution and then taken out and rinsed in distilled water. Chitosan/calcium-alginate microcapsules were prepared in a similar procedure except that Na2HPO4 was not added to the alginate solution prior to droplet formation. All of the procedures were carried out at room temperature and pH 7.0. 2.3. Characterization of Microcapsules. The impregnation characteristics of microcapsules were determined by immersing preweighed microcapsules (the water on the surface was removed by filter paper) to impregnate in 5 mL of deionized water at room temperature, and their weight changes were monitored at specified time intervals. The impregnation ratios were calculated using eq 1.33 In the curves plotted, the complete dissolution of beads has been indicated as a 100% weight change.

impregnation degree (%) ) ((final weight - initial weight)/initial weight) × 100 (1) The morphology of the microcapsule surface was observed by scanning electron microscopy (SEM, XL30, PHILIPS, Holand) with an accelerating voltage of 20 kV, after being freeze dried and gold coated. The surface elemental composition of the capsules was analyzed by X-ray photoelectron spectroscopy (XPS) in a PerkinElmer PHI 1600 ESCA system with a monochromatic Mg KR source and a charge neutralizer. The pore-size distribution of microcapsule shell was determined by nitrogen adsorption-desorption isotherm measurements (BET) performed at 77 K on a CHEMBET-3000 nitrogen adsorption apparatus.

2.4. Encapsulation and Activity Assay of YADH. Chitosan was dissolved in deionized water to obtain a homogeneous solution with a final concentration of 2% (w/v). A 4 mL chitosan solution was mixed with an enzyme stock solution prepared by dissolving 0.96 mg of YADH in 1.0 mL of 0.05 mol/L TrisHCl buffer (pH 7.0) at room temperature. This mixture was added dropwise to a sodium alginate solution (0.8% (w/v)) containing 60 mM Na2HPO4 to produce YADH encapsulated microcapsules as described above. The droplets were left for 10 min in the sodium alginate solution and then taken out and rinsed in distilled water to pH 7.0. Encapsulation efficiency: Capsules were disrupted by cutting to release the YADH inside, and the encapsulation efficiency was determined by the following equation,

encapsulation efficiency ) [YADH]capsule/[YADH]droplet × 100 where [YADH] capsule and [YADH] droplet were the concentrations of YADH in the final capsule and original liquid droplet, respectively. The YADH concentration was determined by the micro-Bradford method. Bradford reagent (2 mL) was added into 2 mL of YADH-containing solution. After incubated for 5 min, the absorbance of the above solution at 595 nm was measured using a UV spectrophotometer (Hitachi U-2800). YADH-catalyzed hydrogenation of formaldehyde to methanol coupling with the oxidation of NADH to NAD+ was used to evaluate the catalytic activity of the free and encapsulated YADH. YADH

HCHO + NADH + H+ 798 CH3OH + NAD+ The standard assay was carried out in a neutral aqueous solution of 10 mM HCHO and 133 µM NADH at different temperatures (20-50 °C). The enzyme activity was determined spectrophotometrically by directly measuring the decrease in absorbance of NADH at 340 nm. Thermal stability:34 The thermal stability of free and encapsulated YADH (including mineralized microcapsules and nonmineralized microcapsules) were evaluated by measuring the enzyme activity at different temperatures. Recycling stability: The encapsulated YADH was filtered after each reaction batch, rinsed with deionized water, and then added to the next reaction cycle. The recycling stabilities of encapsulated YADH were evaluated by measuring the enzyme activity in each successive reaction cycle and expressed by recycling efficiency.

Figure 2. Mineralized chitosan/alginate microcapsules, (a) fresh microcapsule and (b) freeze-dried microcapsule.

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Figure 3. (a) SEM images of surface morphology of a nonmineralized microcapsule. SEM images of the surface morphology of calcium phosphatechitosan/alginate prepared in the presence of (b) 50 mM, (c) 100 mM and (d) 150 mM calcium, and (e) 200 mM CaCl2.

Storage stability: Free and encapsulated YADH were stored at 4 °C for a certain period of time. The storage stability was compared by storage efficiency, defined as the ratio of free or encapsulated enzyme activity after storage to their initial activity. 3. Results and Discussion 3.1 Fabrication and Characterization of Hybrid Microcapsules. 3.1.1. Morphology. Mineralized polysaccharide microcapsules were prepared via a one-step method. The mineralized chitosan/alginate microcapsules were prepared by adding

droplets of Ca2+-containing chitosan solution (2% (w/v)) into phosphate-containing sodium alginate solution (0.8% (w/v)). Because of interfacial complexation of the oppositely charged polysaccharide, a thin alginate film spontaneously formed around chitosan droplets. With increasing time, the microcapsules became much stiffer due to further cross-linking between alginate and Ca2+ ions and the deposition of calcium phosphate (as shown in Figure 1). Microcapsules with an external diameter of ca. 2 mm were obtained. Parts a and b of Figure 2 were the fresh and the freeze-dried microcapsules,

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Figure 4. SEM images of the mineralized microcapsule structure (a) membrane crossection (b) inner structure.

Figure 5. XPS spectra of the mineralized and nonmineralized microcapsule’s surface. It shows that phosphorus was added in the mineral microcapsule surface. This has characteristics of typical XPS spectra obtained for all of the samples studied.

respectively. As seen from Figure 3, in the SEM images of the microcapsule surface, it can be clearly found that the surface of the microcapsules became more compact. The structure of the microcapsules was observed by SEM, and the typical photos were shown in Figure 4. When the CaCl2 concentration was 0.2 M, the thickness of the microcapsule’s shell and the phosphate calcium were ca.15 µm and ca. 7 µm, respectively. The internal surface had a cross-linked structure, whereas the external inorganic layer spread uniformly. In addition, the as-prepared microcapsule showed a hollow core after being freeze-dried. The XPS analysis confirmed the existence of phosphorus element and determined the elemental composition of the microcapsule’s surface as shown in Figure 5. Compared with the nonmineralized microcapsules, the elemental peaks of calcium, sodium, chlorine, carbon, and oxygen had no significant change after they were mineralized, and the elemental peak of phosphorus appeared in the mineralized ones at 136 eV. 3.1.2. Impregnation Property. All of the microcapsules reached impregnation equilibrium in deionized water at 25 °C, and no breakage was found during the testing period. As shown in Figure 6, the impregnation degree of the mineralized microcapsules was 9%, whereas the nonmineralized was 14% at 0.2 M CaCl2. This result can be assigned to the fact that the inorganic layer was rigid enough to effectively protect the microcapsule from impregnation. Furthermore, the impregnation degree of microcapsules was directly related to the CaCl2 concentration. Higher CaCl2 concentration formed not only a denser polymer structure but also a thicker calcium phosphate

Figure 6. Impregnation degree of the mineralized and the nonmineralized microcapsules prepared with various CaCl2 concentrations. Table 1. Leakage and Loading Efficiency of YADH Encapsulated in Nonmineralized and Mineralized Microcapsules kind of microcapsule

leakage of YADH (%)

loading efficiency of YADH (%)

nonmineralized microcapsule mineralized microcapsule

22 11

78 89

layer, which were both beneficial for inhibiting the impregnation of the microcapsules. 3.2. Catalytic Activities and Stabilities of YADH Encapsulated in the Hybrid Microcapsules. 3.2.1 Encapsulation Efficiency. The encapsulation efficiency can be assessed by measuring the YADH concentration in the solution. As shown in Table 1, the encapsulation efficiency was 89% for mineralized microcapsules, whereas it was 78% for nonmineralized microcapsules. Under identical conditions, the mineralized microcapsules retained more YADH molecules than the nonmineralized microcapsules because the external inorganic layer can prevent YADH from leaking during the microcapsules’ formation. 3.2.2. Thermal Stability. The enzyme activity of both free and encapsulated YADH was studied at various temperatures at pH 7.0. Taking the highest activity of YADH under its optimal condition was to be 100%, and the relative activity of YADH was defined as the ratio of its activity to the highest activity. As shown in Figure 7, the optimal temperature for encapsulated YADH was 25 °C, consistent with that of the free YADH. This indicated that the encapsulation carrier did not cause the inactivation of YADH. It was also proven by the circular dichroism (CD) spectrum in Figure 8. The conformation of

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Figure 7. Relative activity of free and encapsulated YADH at different temperatures.

Figure 8. CD spectra of free and encapsulated YADH. The conformation of YADH in microcapsule was similar to that of free YADH.

YADH after encapsulation was quite similar to that of free YADH. The microcapsules with a chitosan liquid core and alginate shell might be regarded as a simplified microenvironment for the YADH. YADH molecules were physically confined in the liquid core, which could provide YADH with sufficient space to move and rotate flexibly as the case in free form. As a result, the contact between YADH and the substrate became much easier than that in pure solid encapsulation matrixes. The encapsulated YADH displayed higher relative activity than its free counterpart under the same reaction temperature. At 50 °C, the free YADH had an activity of ca. 35% relative to the optimum enzyme activity, whereas the encapsulated YADH had a relative activity of ca. 60%. It can be thus deduced that YADH encapsulated in the mineralized microcapsules had a higher activity and better thermal stability over a wide temperature range than free YADH in aqueous solution. To support this viewpoint, differential scanning calorimetry35 (DSC) data was acquired as shown in Figure 9, and the unfolding temperature for the encapsulated YADH was ca. 87.4 °C, whereas the free YADH was ca. 76.5 °C (∆Tm ) ∼10.9 °C). In other words, the encapsulated YADH was in fact more stable against temperature-induced unfolding compared to free YADH in aqueous solution. 3.2.3. Free and Encapsulated Enzyme Activity under Optimal Conditions. Under optimal conditions (25 °C and pH 7.0), the enzyme activity of free and encapsulated YADH in mineralized microcapsules was investigated. As shown in Figure 10, the bioconversion process was monitored by measuring the conversion of NADH with the lapse of time. The reaction rate and the final conversion using encapsulated YADH were a little lower than those using free YADH. The equilibrium conversion using free YADH was obtained at 96.0% in 12 min, whereas that in the case of encapsulated YADH was 92.7% in 14 min.

Figure 9. DSC thermograms of free and encapsulated YADH, (a) free YADH, P1 ) ∼76.5 °C and (b) encapsulated YADH, P2 ) ∼87.4 °C.

Figure 10. NADH conversion with reaction time (25 °C, pH 7.0).

Figure 11. Recycling stability of encapsulated YADH in mineralized and nonmineralized microcapsules.

The enzyme activity unit was defined as the amount of YADH needed to convert 1.0 µmol of NADH/min at 25 °C and pH 7.0. The specific activity of free and encapsulated enzyme was 44.4 U/mg and 41.2 U/mg YADH, respectively. The lower specific activity of encapsulated YADH might be due to the additional diffusion resistance rather than enzyme denaturation. 3.2.4. Recycling Stability. A significant advantage of the enzyme encapsulated in the mineralized microcapsules was their excellent recycling stability. Take the initial activity of encapsulated YADH in microcapsules as 100%. As shown in Figure 11, 52.2% of YADH in mineralized microcapsules could be retained while decrease to 15.2% in nonmineralized ones after successive ten cycles. The lower enzyme activity observed in nonmineralized microcapsules was due to YADH leakage during the multiple soaking, separation, and washing steps employed during the recycling process; whereas, for the YADH encap-

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activity of YADH in mineralized microcapsules was only a little higher than that in nonmineralized ones. This proved that the chitosan core played an important role in storage stability, whereas the inorganic calcium phosphate layer only had a trivial contribution on the storage stability. 4. Conclusions

Figure 12. Pore-size distribution of the calcium phosphate-mineralized microcapsule’s shell.

This study described a facile procedure for preparing a mineralized polysaccharide capsule as a novel and efficient enzyme encapsulation carrier. This millimeter-sized capsule was of a typical core-shell structure: the biocompatible chitosan liquid core was responsible for accommodating the suitable microenvironment for YADH, and the alginate-templated calcium phosphate outer shell ensured the facile accessibility of encapsulated YADH for substrates and effectively prevented YADH from leaching out. Because of the appropriate combination of hydrophilic polymer and mechanically stable calcium phosphate, the impregnation stability, thermal stability, recycling stability, and storage stability of the enzyme-containing hybrid microcapsules were significantly improved. Further work in this area is ongoing in our laboratories because we believe that calcium phosphate-mineralized alginate/chitosan capsules will find promising applications in pharmacy, biomedical, biotechnology, and biosensor fields. Acknowledgment

Figure 13. Storage stability of free and encapsulated YADH.

sulated in mineralized microcapsules, YADH leakage was prevented during recycling process and the recycling stability was increased substantially. The pore size of the semipermeable microcapsule shell should meet two requirements: it must be big enough for the substrates and products to transport in and out freely but small enough to prevent the enzyme from leaking. The average pore diameter of the mineralized microcapsule shells was determined by BET. As the data shows in Figure 12, the pore diameter of the mineralized shell was around 3 nm. Because the dynamic diameter of the substrates molecule was less than 1 nm and the dynamic diameter of YADH was about 7 nm, it would be naturally concluded that the calcium phosphate shell can prevent YADH from leaking but be facile for the diffusion of substrates and products. 3.2.5. Storage Stability. The storage stability of encapsulated YADH was also tested. YADH encapsulated in both mineralized and nonmineralized microcapsules maintained a relative activity of more than 50% after 35 days of storage (Figure 13). In contrast, free YADH lost its entire activity within 30 days. It was reasonably believed that the encapsulated YADH would exhibit a distinct advantage over free enzyme in long-time storage, owing to unique encapsulation technology and a special microenvironment provided by the biomimetic microcapsule. For the microcapsule, the liquid chitosan core carried a net positive charge under the neutral pH environment. Therefore, the conformational transition of YADH from the folded to the unfolded state that may likely result in denaturalization can be effectively inhibited by the electrostatic interaction between the negative YADH and positive chitosan molecules. In addition, the biocompatible chitosan was able to help the YADH to effectively avoid the unfavorable influence possibly rising from the outside storage environment. Simultaneously, the relative

Financial support from the National Natural Science Foundation of China (Grant No. 20576096), the Cross-Century Talent Raising Program of Ministry of Education of China, the program for Changjiang Scholars and Innovative Research Team in University (PCSIRT), and the Program of Introducing Talents of Discipline to Universities (No. B06006) are greatly appreciated. Literature Cited (1) Mitzi, D. B.; Medeiros, D. R.; DeHaven, P. W. Low-temperature melt processing of organic-inorganic hybrid films. Chem. Mater. 2002, 14, 2839-2841. (2) Mitzi, D. B.; Prikas, M. T.; Chondroudis, K. Thin film deposition of organic-inorganic hybrid materials using a single source thermal ablation technique. Chem. Mater. 1999, 11, 542-544. (3) Shchipunov, Yu. A.; Burtseva, Yu. V.; Karpenko, T. Yu.; Shevchenko, N. M.; Zvyagintseva, T. N. Highly efficient immobilization of endo1,3-β-d-glucanases (laminarinases) from marine mollusks in novel hybrid polysaccharidesilica nanocomposites with regulated composition. J. Mol. Catal., B 2006, 40, 16-23. (4) Cao, L. Q. Immobilized enzymes: Science or art? Curr. Opin. Chem. Biol. 2005, 9, 217-226. (5) Cao, L. Q.; Langen, L. V.; Sheldon, R. A. Immobilized enzymes: Carrier-bound or carrier free? Curr. Opin. Biotechnol. 2003, 14, 387-394. (6) Bornscheuer, U. T. Immobilizing Enzyme: How to create more suitable biocatalysts. Angew. Chem., Int. Ed. 2003, 42, 3336-3337. (7) Wong, Y. D.; Sun, D. D.; Lai, D. Value-added utilization of recycled concrete in hot-mix asphalt. Waste Manage. 2007, 27, 294-301. (8) Guglielmi, M.; Brusatin, G.; Giustina, G. D. Hybrid glass-like films through sol-gel techniques. J. Non-Cryst. Solids. 2007, 353, 1681-1687. (9) Hall, S. R.; Davis. S. A.; Mann, S. Cocondensation of organosilica hybrid shells on nanoparticle templates: A directed synthetic route to functionalized core-shell colloids. Langmiur 2000, 16, 1454-1456. (10) Molvinger, K.; Quignard, F.; Brunel, D.; Boissiere, M.; Devoisselle, J. M. Porous chitosan-silica hybrid microspheres as a potential catalyst. Chem. Mater. 2004, 16, 3367-3372. (11) Zhou, J.; Chen, M.; Qiao, X. G.; Wu, L. M. Facile preparation method of SiO2/PS/TiO2 multilayer core-shell Hybrid microspheres. Langmiur 2006, 22, 10175-10179. (12) Regi, M. V.; Salinas, A. J.; Castellanos, J. R.; Gonza´lez-Calbe, J. M. Nanostructure of bioactive sol-gel glasses and organic-inorganic hybrids. Chem. Mater. 2005, 17, 1874-1879.

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ReceiVed for reView September 29, 2007 ReVised manuscript receiVed January 28, 2008 Accepted February 8, 2008 IE071315R