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Enhancement of Enzymatic Activity Using Microfabricated Poly(#–caprolactone)/Silica Hybrid Microspheres with Hierarchically Porous Architecture Yimei Fan, Xiaodong Cao, Tao Hu, Xiaoguang Lin, Hua Dong, and Xuenong Zou J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.5b12269 • Publication Date (Web): 29 Jan 2016 Downloaded from http://pubs.acs.org on February 2, 2016

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The Journal of Physical Chemistry

Enhancement of Enzymatic Activity Using Microfabricated Poly(ε–caprolactone)/Silica Hybrid Microspheres with Hierarchically Porous Architecture Yimei Fan1, 2, Xiaodong Cao1, 2, 3,*, Tao Hu1, 2, Xiaoguang Lin1, 2, Hua Dong1, 2, *, Xuenong Zou4

1. Department of Biomedical Engineering, School of Materials Science and Engineering, South China University of Technology, Guangzhou, 510006, China 2. National Engineering Research Center for Tissue Restoration and Reconstruction (NERC-TRR), Guangzhou, 510006, China 3. Guangdong Province Key Laboratory of Biomedical Engineering, South China University of Technology, Guangzhou, 510641, China 4. Guangdong Province Key Laboratory of Orthopedics and Traumatology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510075, China

*Corresponding authors: [email protected] (X.D. Cao) and [email protected] (H. Dong)

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ABSTRACT

In this paper, we present a novel and facile microfluidic method to fabricate hierarchically porous poly(ε–caprolactone)/silica (PCL/SiO2) hybrid microspheres and further investigate in detail their performance as enzyme carriers by three famous proteins and enzymes. Due to the synergy effect between sol-gel process and solvent extraction in micro-droplets, hierarchically porous architecture can be formed in situ without the use of porogens and templates. More importantly, the surface porosity or the specific surface area of such microspheres can be precisely tuned via adjusting the hydrolysis/condensation rate by ammonia catalyst and thus the competition between the two above-mentioned processes. Fluorescein isothiocyanate-bovine serum albumin (FITC-BSA), alcohol dehydrogenase (ADH) and superoxide dismutase (SOD) are immobilized via either physical adsorption or covalent binding to evaluate the performance of hierarchically porous microspheres as enzyme carriers. All the qualitative and quantitative data including fluorescence images, enzymatic activity, immobilization yield and activity yield prove that enzymes covalently immobilized on hierarchically porous microspheres exhibit the optimal immobilization capacity, enzymatic activity, stability and reusability, which shows very promising application of such microspheres in enzymatic catalysis.

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Introduction As an environmentally friendly alternative to traditional chemical catalysts, enzymes exhibit several unique advantages such as high selectivity, high efficiency and mild operational condition, which are of significance for a wide range of applications including the synthesis of pharmaceuticals and fine chemicals, analysis of chemical and biological substances, food processing, biofuel production, etc. 1-3 Unfortunately, their poor stability and loss of catalytic activity even after one cycle are one of the greatest obstacles for the use of free-standing enzymes in practical processes,4-5 and thus have stimulated intense studies aimed at the improvement of their properties 6. Among state-of-the-art methods that have been developed to overcome these drawbacks, enzyme immobilization on solid substrates through physical adsorption or chemical modification is the most extensively applied one 7-8. Tremendous materials have been tested as enzyme carriers, in which porous supports have received widespread attention due to their high surface area and ability to encapsulate enzymes within the pores to provide a more stable microenvironment.9 In particular, hierarchically porous materials, or namely, mesoporous materials with macroporous structures are ideal carriers because the textural mesopores and interconnected pore systems of the macrostructures can efficiently transport substances to framework binding sites.10 Nowadays, there are numerous techniques to synthesize hierarchically porous membranes and microspheres with controllable dimensions from nanoscale to macroscale.11-13 Typical strategies are to make use of pore-forming agents with well-defined size and structure like gas foaming, colloidal crystals,

18,19

14

salt leaching,

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acid extraction,

16

freeze drying,

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liquid or

etc. However, these methods in general need additional templates or

porogens to achieve porous structures, indicative of partial residual after complicated post-treatment and possible deactivation of immobilized enzymes. Therefore, exploration of new porous carriers fabricated in a facile, efficient and free of pore-forming agent manner is still highly necessary for enzymatic catalysis. In this paper, we demonstrate a simple and robust droplet-based microfluidic approach to prepare hierarchically porous inorganic/organic hybrid microspheres and further evaluate in detail their performance as enzyme carriers by physically and chemically immobilizing three different proteins and enzymes (Scheme 1). The superiority of this new strategy lies in the formation of hierarchically porous structures by a synergy effect between sol-gel process (hydrolysis and 3



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condensation of a silane precursor) and solvent extraction in micro-droplets, which not only generates homogeneous inorganic functional component (silica) in the microspheres but also avoids addition of any template or porogen. Instead of sole silicate component in classical enzyme carrier,

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, biocompatible polycaprolactone (PCL) is introduced to enhance the affinity of

enzymes to the microsphere matrix and thus improve enzyme immobilization amount. Specifically, influential factors affecting the surface porosity of hybrid microspheres during the fabrication process and parameters characterizing enzyme immobilization such as activity of immobilized enzyme, immobilization yield, activity yield and reusability are investigated. Our results show that hierarchically porous PCL/SiO2 hybrid microspheres `are very promising carriers for enzymatic catalysis.

Experimental Materials and reagents PCL (Mw = 80 kDa) was purchased from Sigma (USA). Poly(vinyl alcohol) (PVA, 87-89% hydrolyzed, Mw = 88 kDa), tetraethylorthosilicate (TEOS), (3-Aminopropyl)triethoxysilane (APTES) (purity ≥ 99%), nitro-blue tetrazolium chloride (NBT), ethylene diamine tetraacetic acid (EDTA) and riboflavin were bought from Aladdin Chemistry (Shanghai, China). Glutaraldehyde (50%)

was

obtained

from

Tianjing

Damao

Chemical

Co.

Ltd

(Tianjin,

China).

Polydimethylsiloxane (PDMS) (Sylgard 184) was purchased from Dow Corning Company (USA). Fluorescein isothiocyanate-bovine serum albumin (FITC-BSA) was purchased from Bioss Co. Ltd (Beijing, China), while alcohol dehydrogenase (ADH), superoxide dismutase (SOD), nicotinamide adenine dinucleotide (NAD) and methionine were all bought from Qiyun Biotechnology Co. Ltd (Guangzhou, China). All the chemicals were used as received without further purification.

Microfluidic fabrication and characterization of porous PCL/SiO2 hybrid microspheres Droplet-based microfluidic devices were fabricated using the method described in the literature

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. In brief, negative photoresist was first spin-coated on a clean silicon wafer. After

baking at 80oC for 10 min and 150oC for 5 min, the resist was exposed to UV light through a photo-mask and then developed in developer solution. Mixture of PDMS base and curing agent (10:1 w/w) was poured onto the silicon wafer, degassed by vacuum oven and cured on a hotplate 4


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at 80oC for 6h. The PDMS replica was subsequently peeled off and sealed with a glass slide via O2 plasma. Surface treatment was conducted to improve the hydrophilicity of microchannels by injecting PVA/glycerol (2/5 wt%) aqueous solution and curing for 1h. In a typical process, 40 mg/mL of PCL and TEOS dissolved in dichloromethane was used as the dispersed phase, whilst mixture of PVA (2 wt%) and ammonia solution was used as the continuous phase. The two phases were introduced into the microfluidic device using syringe pumps (Cole-Parmer, USA), and droplets were generated continuously at the junction of the microchannels by adjusting the flow rates of the continuous and dispersed phases. The as-formed droplets were collected in 2 wt% of PVA aqueous solution. After settled at room temperature for 24 h, the solidified microspheres were centrifuged, washed with DI water and dried in a freeze-drier (Lyophilizer, VIRTIS, USA). The surface morphology of PCL/SiO2 hybrid microspheres was characterized using a field-emission scanning electron microscope (FE-SEM, NOVA 430, Netherlands). The chemical composition of the microsphere surface was analyzed both with an electron probe micro-analyzer (EPMA, EPMA-1610, Shimadzu, Japan) and with an attenuated total reflection Fourier transform infrared spectroscope (ATR-FTIR, Nexus Por Euro, USA). In order to figure out the actual content of inorganic components in PCL/SiO2 hybrid microspheres, thermogravimetric and differential scanning calorimetry (TG-DSC, Diamond, Germany) were performed under an oxygen atmosphere. The specific surface area, pore diameter and size distribution were measured using Brunauer-Emmett-Teller (BET, Nova4200e, Quantachrome, USA).

Surface modification of PCL/SiO2 hybrid microspheres Prior to surface modification, heat treatment was carried out to remove residual water on the microsphere surface. APTES, a silane coupling agent with -NH2, was then used to react with SiO2 on the microsphere surface. Specifically, 0.2 g of microspheres were dispersed in 40 mL of ethanol solution containing 0.02 g APTES and then stirred at 40℃ for 6 h. The modified microspheres were filtered, washed to remove unreacted APTES and finally dried for 48 h.

Immobilization of FITC-BSA and enzymes APTES-modified microspheres were activated with glutaraldehyde in Tris-HCl buffer (0.02 M, pH 8.0), which could rapidly form stable imide bonds with the amino groups of the proteins. 5



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The mixture was constantly stirred for 2 h and rinsed to remove the unbound glutaraldehyde. 20 µL of FITC-BSA solution were mixed with 0.08 g of activated microspheres in Tris-HCl buffer, followed by gentle shake at ambient temperature in darkness for 6 hours. Subsequently, FITC-BSA immobilized microspheres were filtered and washed with Tris-HCl buffer for 3 times, and their fluorescence intensity was measured by confocal laser scanning microscope (CLSM, ZeissLsm-510, Japan). Similarly, 0.08 g of activated PCL/SiO2 microspheres was suspended in buffer solution. 0.2 mL of ADH or SOD was then added. After gently stirred at room temperature for 10 h, the mixture was centrifuged and washed three times to remove the unbound enzyme.

Activity and reusability assay of immobilized enzymes Enzymatic activity of ADH was determined by quantification of NADH formation, as measured by the increase in absorbance intensity at 340 nm using UV-vis spectrophotometer (Tu-1901, China). 30 µL of ADH (0.08 mg/mL) was added into 2 mL of Tris-HCl buffer (0.05M, pH 8.8), and the enzymatic reaction was initiated by addition of 40 µL of ethanol (30 mM) and 10µL of NAD (1.5 mM), and the whole solution was kept at 35oC for 5 min before immersed in boiling water for 1 min to terminate the reaction. Control group used deionized water rather than ethanol. Alternatively, the activity of immobilized ADH was measured by addition of ADH-immobilized microspheres. ADH activity was expressed as the amount of ADH required to hydrolyze 1.0 mol of ethanol per minute. The activities of free and immobilized SOD were determined by measuring its ability to inhibit the reduction of NBT. Solution containing 0.2 mL of NBT (0.075 mM), EDTA (10 nM), methionine (13 mM), 20 µL of SOD (0.04 mg/mL) was mixed with 2 mL of PBS (0.05M, pH 7.8). 0.2 mL of riboflavin (2 µM) was added at the end under the dark condition. Each sample was shaken and then illuminated by lamp for 15 min while the control group was placed in the dark. The absorbance intensity at 560 nm was measured to calculate the activity of free and immobilized SOD. SOD activity was expressed as the amount of SOD required to inhibit the reduction of NBT (inhibit the formation of formazan) by 50% per minute. The reusability of immobilized ADH and SOD was investigated by determining their remaining activity after each cycle. The activity of immobilized enzymes in the first cycle was defined as 100% and relative activity was calculated for the following cycles. 6


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All the relevant data of the activity and reusability assay were collected within the optimal temperature range of ADH and SOD enzymes (35-40oC).

Results and discussion Fabrication and characterization of hierarchically porous PCL/SiO2 microspheres In this study, droplet-based microfluidics was employed to fabricate hierarchically porous PCL/SiO2 hybrid microspheres due to its ability to precisely control the droplet/particle size and size distribution.25, 26 Right after the generation of PCL/TEOS droplets in the microchannel, the organic solvent in the dispersed phase and water in the continuous phase diffuse spontaneously toward the opposite direction, leading to in situ hydrolysis and condensation of TEOS on the droplet surface. As a result of the synergy effect between sol-gel process and solvent extraction, PCL/SiO2 hybrid microspheres with controllable surface porosity can be harvested. Figure 1 compares pure PCL and PCL/SiO2 hybrid microspheres fabricated using the same device but different conditions. It is also obvious that all the microspheres are of excellent monodispersity, uniform size and narrow size distribution (Figure 1)，but their surface morphologies vary dramatically. The pure PCL microspheres shown in Figure 1a display a smooth surface, indicating that the solvent extraction and PCL itself cannot induce surface porosity. Similarly, the surfaces of PCL/SiO2 hybrid microspheres prepared without addition of ammonia in the aqueous phase (the continuous phase and/or the collection solution) are quite smooth. Moreover, the variation in the mass ratio of TEOS to PCL doesn’t cause obvious change in surface morphology of hybrid microspheres (Figure S1, Figure S2 a, d, g, supporting information, SI). However, once ammonia, a catalyst for silane hydrolysis, was added, the as-prepared microspheres show hierarchically porous configurations and the surface porosity can be enhanced by increasing the ammonia concentration (Figure 1, c-d). To further confirm the role of ammonia in the formation of hierarchically porous structure, the hydrolysates SiO2 and ethanol, rather than TEOS, were added into the dispersed phase respectively. The results reveal that the presence of SiO2 and ethanol cannot form pores on the microsphere surface during the fabrication process (Figure S3, SI). Besides, the porous PCL/SiO2 hybrid microspheres obtained using sodium hydroxide as catalyst exclude the possible influence from ammonium (Figure S4, SI). In our opinion, the hierarchically porous structure can be attributed to the competition between sol-gel process and solvent 7



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extraction. When the hydrolysis and condensation of TEOS is much slower than solvent extraction, microspheres with smooth surface can be obtained. In contrast, if the sol-gel process is accelerated under the catalysis of ammonia and becomes much faster in comparison to solvent extraction, hierarchically porous microspheres are produced with high yield. It should be noted that ammonia in the continuous phase is more effective than that in the collection solution (Figure 2), implying the porous structure of microsphere is formed immediately after the generation of PCL/TEOS droplets. Interestingly, in the presence of ammonia, altering the mass ratio between TEOS and PCL can cause the change in surface porosity (Figure S2, SI). A reasonable explanation is that the activity of water in alkaline medium is higher than that of TEOS and thus the hydrolysis/condensation rate is dominated by the TEOS concentration. The in-situ fabrication of hierarchically porous PCL/SiO2 hybrid microspheres not only avoids the use of porogens/templates that require complicated post-treatment for removal, but also ensures the uniform distribution of silica in the PCL matrix. Herein EPMA, FTIR and TG-DSC measurements were performed to identify the chemical composition of the hybrid particles with the results summarized in Figure 3. EPMA analysis compares pure PCL, pure SiO2 particles fabricated by hydrolysis of TEOS, and PCL/SiO2 hybrid microspheres that were all prepared in the same microfluidic device (Figure 3a). It is clear that Si element is absent from pure PCL microspheres but present on TEOS hydrolysate and PCL/SiO2 hybrid microsphere, implying that Si in PCL/SiO2 hybrid microsphere is originated from TEOS hydrolysis. C and O elements detected on the pure SiO2 particles can be ascribed to residual PVA surfactant. ATR-FTIR profiles further confirm such observation. As shown in Figure 3b, the obvious absorption peak at 1098 cm-1 in curve d’ can be attributed to the bending vibration of Si-O, and the broad peak at 3100-3700 cm-1 is caused by H2O. Meanwhile, the broad and strong absorption bands at ca. 2800-3000 cm-1 and 700-1500 cm-1 in curve e’ can be assigned to the bending and stretching vibration of C-H, respectively. Another characteristic absorption peak at 1730 cm-1 is due to the stretching vibration of C=O. Evidently, peaks of PCL and SiO2 can be both detected on hybrid microsphere surface (curve f’), and this phenomenon proves that the PCL/SiO2 hybrid microsphere surface is composed of PCL and SiO2. Figure 3c shows the TG-DSC data of PCL/SiO2 hybrid microspheres. The exothermic peaks at 245oC and 382oC refer to the dehydration of Si-OH and combustion of PCL. At 441°C and 481°C, there are two small 8


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exothermic peaks, which are probably resulted from the further oxidation of carbonized residues coming from the incomplete combustion of PCL 27. The mass percent of incombustible SiO2 in the hybrid microsphere is ca. 7.8wt%. In addition, BET test shows that the specific surface areas are 2.741 m2/g for pure PCL microspheres, 3.136 m2/g for smooth PCL/SiO2 hybrid microspheres, 67.525 m2/g for dimpled PCL/SiO2 hybrid microspheres, 333.698 m2/g for PCL/SiO2 hybrid microspheres with deep pores.

Protein immobilization via physical adsorption and covalent binding Considering the high affinity of biocompatible PCL to enzyme and the high dispersion of SiO2 in PCL/SiO2 hybrid microspheres that can be decorated to carry functional groups, enzyme immobilization was realized through either physical adsorption or covalent binding.

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In order

to evaluate the immobilization capability of the above-mentioned microspheres in a straightforward way, FITC-BSA was first immobilized and observed using CLSM. It can be found from the fluorescence images (Figure 4) that pure PCL microspheres display very weak immobilization capacity no matter for physical adsorption or covalent binding (Figure 4a). In contrast, although the specific surface area is very similar, smooth PCL/SiO2 hybrid microspheres exhibit enhanced BSA immobilization especially using covalent binding mode (Figure 4b), which is mainly due to the existence of SiO2 on the microsphere surface. This tendency is further reinforced when the specific surface area of PCL/SiO2 hybrid microspheres increases (Figure 4c, 3d). Compared with physical adsorption, chemical binding benefits more remarkably the BSA immobilization, and significant difference (P

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