Enzyme-Immobilized Chitosan Nanoparticles as Environmentally

Article Cite This: Biomacromolecules XXXX, XXX, XXX−XXX

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Enzyme-Immobilized Chitosan Nanoparticles as Environmentally Friendly and Highly Effective Antimicrobial Agents Kyung-Min Yeon,† Jisung You,‡ Manab Deb Adhikari,‡ Sung-Gil Hong,‡ Inseon Lee,‡ Han Sol Kim,‡ Li Na Kim,‡ Jahyun Nam,‡ Seok-Joon Kwon,§ Moon Il Kim,∥ Warayuth Sajomsang,⊥ Jonathan S. Dordick,*,§ and Jungbae Kim*,‡ †

Construction Technology Team, Samsung C&T Corporation, Gyeonggi-Do 13530, Korea Department of Chemical and Biological Engineering, Korea University, Seoul 02841, Korea § Department of Chemical and Biological Engineering, and Center for Biotechnology & Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180, United States ∥ Department of BioNano Technology, Gachon University, Gyeonggi-Do 13120, Korea ⊥ National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA), Klong Luang, Pathum Thani 12120, Thailand

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S Supporting Information *

ABSTRACT: Highly effective and minimally toxic antimicrobial agents have been prepared by immobilizing glucose oxidase (GOx) onto biocompatible chitosan nanoparticles (CS-NPs). CS-NPs were prepared via ionotropic gelation and used for the immobilization of GOx via approaches of covalent attachment (CA), enzyme coating (EC), enzyme precipitate coating (EPC), and magnetic nanoparticle-incorporated EPC (Mag-EPC). EPC represents an approach consisting of enzyme covalent attachment, precipitation, and cross-linking, with CA and EC being control samples while Mag-EPC was prepared by mixing magnetic nanoparticles (Mag) with enzymes during the preparation of EPC. The GOx activities of CA, EC, EPC, and Mag-EPC were 8.57, 17.7, 219, and 247 units/mg CS-NPs, respectively, representing 26 and 12 times higher activity of EPC than those of CA and EC, respectively. EPC improved the activity and stability of GOx and led to good dispersion of CS-NPs, while MagEPC enabled facile magnetic separation. To demonstrate the expandability of the EPC approach to other enzymes, bovine carbonic anhydrase was also employed to prepare EPC and Mag-EPC samples for their characterizations. In the presence of glucose, EPC of GOx generated H2O2 in situ, which effectively inhibited the proliferation of Staphylococcus aureus in both suspended cultures and biofilms, thereby demonstrating the potential of EPC-GOx as environmentally friendly and highly effective antimicrobial materials.

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INTRODUCTION Microbial contamination in myriad environments threatens human health1,2 and causes irreversible deterioration of materials and manufacturing processes.3 Microbial contamination often takes the form of biofilms, in which aggregated bacterial cells are embedded within a self-secreted matrix composed of extracellular polymeric substances (EPS).1,2 Antimicrobial agents have been used extensively to control this biofilm formation. In most cases, however, harsh chemicals such as sodium hydroxide or sodium hypochlorite have been © XXXX American Chemical Society

employed, which are toxic, can lead to infrastructure corrosion, and can be highly disruptive of natural ecosystems.4 In addition, biofilms have a high tolerance to external chemical agents as a result of the diffusional barrier provided by the EPS, raising the issue of overdosing. Therefore, there remains a need to develop highly effective and less-toxic antimicrobial agents. Received: January 30, 2019 Revised: May 1, 2019

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DOI: 10.1021/acs.biomac.9b00152 Biomacromolecules XXXX, XXX, XXX−XXX

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Figure 1. Schematic representation of enzyme immobilization onto CS-NPs via four different approaches, which are CA, EC, EPC, and Mag-EPC.

magnetic nanoparticle-incorporated EPC (Mag-EPC). The EPC approach consists of three sequential steps including covalent enzyme attachment (CA), enzyme precipitation, and cross-linking. EC serves as a control without the step of enzyme precipitation, while Mag-EPC is prepared by adding amine-functionalized magnetic nanoparticles during the enzyme precipitation step of EPC. Both EPC and Mag-EPC greatly improved the stability as well as loading and activity of GOx on CS-NPs, consistent with previous nanobiocatalytic studies.37−40 As another model enzyme, bovine carbonic anhydrase (bCA), which catalyzes the conversion of carbon dioxide to bicarbonate, was also immobilized on CS-NPs to demonstrate the extendibility of the EPC method to another enzyme. Finally, GOx-immobilized CS-NPs employed the antibacterial activities against Staphylococcus aureus, thereby demonstrating the potential of GOx-immobilized CS-NPs to serve as highly effective antimicrobial agents.

Enzymes have gathered growing attention as an environmentally friendly alternative to conventional antimicrobials.5−9 Enzymes can effectively control biofilm formation under mild conditions via degradation of adhesive biopolymers, disruption of biofilm matrices, biocide generation, and interference with bacterial communication.5 In particular, glucose oxidase (GOx) has been used in the food industry as an antibacterial agent because of its ability to catalyze the synthesis of hydrogen peroxide (H2O2) from glucose.10,11 Chitosan is one of the most abundant natural polymers on Earth and is highly biocompatible and biodegradable, which enables its ubiquitous use in the environment and biomedicine, including wastewater treatment,12−15 controlled drug delivery,16−18 gene therapy,19,20 tissue engineering,21,22 and wound healing.23−25 The primary amino groups in chitosan enable facile conjugation of biomolecules, and thus chitosan has been used as a common matrix for enzyme immobilization.26−31 These attributes have led to chitosan fabricated at the nanoscale, for example, via ionotropic gelation of multivalent anion and cationic chitosan.32 However, chitosan nanoparticles (CS-NPs) synthesized via ionotropic gelation generally result in spontaneous aggregation in aqueous solution and irreversible fusion with one another upon drying.33,34 This aggregation complicates the handling of CS-NPs and increases mass-transfer resistance, both of which restrict the practical applications of CS-NPs. To the best of our knowledge, only a few methods have been proposed to prevent aggregation of CS-NPs. These methods include introducing a stabilizer that can neutralize the chitosan amino groups35 or modifying the amines with hydrophobic groups.36 However, these approaches inactivate the primary amines, which dramatically reduce the ability of biomolecules to be conjugated to CS-NPs. In the present work, we developed highly efficient, ecofriendly, and reusable antimicrobial agents using enzymeimmobilized CS-NPs, which exhibited excellent dispersion in aqueous solution and retained high enzyme activity and stability. CS-NPs were prepared via ionotropic gelation, and GOx was subsequently immobilized via four distinct approaches involving covalent attachment (CA), enzyme coating (EC), enzyme precipitate coating (EPC), and

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EXPERIMENTAL SECTION

Materials. Chitosan (Mw: 50 000−190 000 Da, degree of deacetylation: 75−85%, viscosity: 20−300 cP, 1 wt % in 1% acetic acid), GOx from Aspergillus niger, bCA from bovine erythrocytes, bovine serum albumin (BSA), sodium tripolyphosphate (TPP), horseradish peroxidase, ß-D-glucose, 3,3′,5,5′-tetramethylbenzidine (TMB), 4-nitrophenyl acetate (NPA), acetic acid, glutaraldehyde (GA) solution (25%), TWEEN 20, ammonium sulfate, carboxyfluorescein diacetate succinimidyl ester (cFDA-SE), and agar were purchased from Sigma (St. Louis, MO, USA). Aminosilane-coated magnetic nanoparticles (fluidMAG-Amine, 50 nm, 25 mg/mL) were purchased from Chemicell (Berlin, Germany). The Petrifilm was purchased from 3 M (St. Paul, MN, USA). The cotton gauze was purchased from Sangkong Pharm (Yongin, Rep. of Korea). Synthesis of CS-NPs. CS-NPs were synthesized via ionotropic gelation of chitosan with TPP anions41 as graphically depicted in Figure S1. Briefly, 10 mg of chitosan was dissolved in the 5 mL of acetic acid (1%, v/v). The insoluble residue in the chitosan solution was removed using Whatman filter paper #1. Simultaneously, TPP solution (0.06%, w/v) was prepared in distilled water. TWEEN 20 solution was added to both chitosan and TPP solution at a final concentration of 0.5% (v/v) as a surfactant to prevent aggregation during ionotropic gelation. TPP solution was added dropwise (1 mL/ min) to the chitosan solution under magnetic stirring (500 rpm) at B


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Biomacromolecules room temperature for 15 min. Then, 0.4 mL of GA solution (25%, v/ v) was added to the chitosan−TPP mixture and stirred for 10 min (500 rpm) by using a magnetic stirrer. The final solution pH was adjusted to 6.0 by adding 1 M NaOH solution. After overnight incubation at 4 °C on a rocking shaker (50 rpm), the solution was centrifuged at 13 000 rpm for 1 h and then the supernatant was decanted. The prepared CS-NPs were finally suspended into 10 mL of distilled water and stored at 4 °C until use. Immobilization of Enzymes on CS-NPs. GOx and bCA were immobilized onto CS-NPs via CA, EC, EPC, and Mag-EPC approaches (Figure 1). CS-NPs (2 mL, 1 mg/mL) were washed twice with distilled water and then resuspended in 0.5 mL of 100 mM sodium phosphate buffer (PB) at pH 7.0 and 7.6 for GOx and bCA, respectively. CA was prepared by mixing 0.5 mL of CS-NP solution with the same volume of enzyme solution (20 mg/mL). Then, 1.5 mL of PB was added to adjust the final product volume of 2.5 mL. For EC, 0.1 mL of GA solution was added to CA at a final concentration of 0.5% (v/v) and the final volume was achieved by adding PB. For the preparation of EPC, 1.4 mL of ammonium sulfate solution was added at a final concentration of 50% (w/v) to the CA. In the case of Mag-EPC, magnetic nanoparticles (25 mg/mL) were additionally introduced at a final concentration of 12.5 mg/mL. After shaking EPC and Mag-EPC samples at room temperature (200 rpm, 30 min), 0.1 mL of GA solution was added to the mixture at a final concentration of 0.5% (v/v). All CS-NP preparations were incubated on a rocking shaker at 4 °C (50 rpm) for 17 h. After washing three times with PB, 1.5 mL of TrisHCl buffer (100 mM, pH 7.4) was added to all samples and incubated for 30 min to cap the unreacted aldehyde groups of GA. Finally, all the samples were excessively washed with PB and stored at 4 °C until use. Particle Characterization. Scanning electron microscopy (SEM) was performed to examine the morphology of enzyme-immobilized CS-NPs. Samples were excessively washed by deionized water to remove salts and freeze-dried for 24 h. After platinum coating, the samples were analyzed at an accelerating voltage of 15 kV. The particle diameter in the SEM image was quantitatively determined using ImageJ software.42 The particle size distribution and zeta potential of CS-NP preparations were measured using ELSZ-1000 (Otsuka Co., Tokyo, Japan). The samples were dispersed into the deionized water with a dilution ratio of 1:20. Enzyme Activity and Stability. The activity of GOx was determined by measuring TMB oxidation according to a standard procedure.43 In brief, a reaction cocktail was prepared by mixing peroxidase (3.79 mg/mL), TMB (0.576 mg/mL), and glucose (110 mg/mL) in 100 mM PB (pH 7.0). Then, 0.9 mL of reaction cocktail was mixed with 100 μL of immobilized enzyme sample. The oxidation of TMB was measured by the increase of absorbance at 655 nm using a UV−vis spectrophotometer (UV-2450, Shimadzu, Japan). The activity of bCA was measured by the hydrolysis of NPA. NPA solution (60 mM) was prepared in acetonitrile. After mixing 50 μL of NPA solution with 850 μL of PB, the enzyme sample (100 μL) was added to the NPA solution (900 μL) to initiate the enzyme reaction. The increase of absorbance at 348 nm was measured spectrophotometrically. These experiments were conducted in triplicate. Enzyme stability was determined by measuring the residual enzyme activity at predetermined time points after incubation of sample at room temperature. The relative activity was calculated from the ratio of residual activity at each time point to the initial activity of each sample. Antibacterial Assay for Suspended Growth. Antibacterial tests were carried out by turbidity method and colony-forming unit (cfu) assay. S. aureus (KCTC 3881) was used as a model bacterial strain. The cells were propagated in nutrient broth (NB) at 37 °C under shaking at 180 rpm for 12 h. To observe the inhibition of bacterial growth, 50 μL of S. aureus culture was inoculated into 5 mL of fresh NB with glucose (0.5%, w/v) and GOx-immobilized CS-NPs (200 ng CS-NPs/mL). Each sample was incubated at 37 °C on the shaker (180 rpm) for 6 h. Inhibition of bacterial growth was evaluated by

measuring the increase of absorbance at 600 nm using a microplate reader (Tecan Trading AG, Männedorf, Switzerland). For cfu assay, 3 mL of bacterial suspension (106 cfu/mL) in phosphate-buffered saline (PBS) was prepared with glucose (0.5%, w/ v) and GOx-immobilized CS-NPs (50 ng CS-NPs/mL). Each sample was incubated at 37 °C on the shaker (100 rpm) for 3 h. For control sample, only PBS solution was added and the sample was incubated in the same condition. Aliquots were sampled at 0, 1, 2, and 3 h. The diluted solution (1 mL) from each aliquot was drawn from the tube and spread on a Petrifilm. The cell number represented by cfu was counted after the incubation at 37 °C for 24 h. The minimum bactericidal concentrations (MBCs) of EPC-GOx and Mag-EPC-GOx were determined by treating the 3 mL of bacterial suspension (106 cfu/mL) with 0−50 ng of CS-NPs/mL and incubating at 37 °C on the shaker (100 rpm) for 3 h. The experiments were conducted in triplicate. cFDA-SE Leakage Assay. Overnight grown cultures of S. aureus (1 mL) were harvested by centrifugation at 8000 rpm for 3 min. The cell pellet was washed twice with sterile PBS and labeled with cFDASE (a final concentration of 50 μM) at 37 °C for 20 min. After incubation, the cell pellets were centrifuged down and washed twice with sterile PBS to remove excess cFDA-SE molecules. The labeled cells (approximately 106 cfu/mL) were treated with GOx-immobilized CS-NPs at 37 °C at 180 rpm for 3 h. The dose of GOx-immobilized CS-NPs was set to 10 μL. For control sample, only PBS solution was added to labeled cells and incubated under the same condition. Subsequently, at each time point, all the samples were centrifuged at 8000 rpm for 10 min. Leakage of carboxyfluorescein from cells was determined by measuring the fluorescence of cell-free supernatant at an emission wavelength of 518 nm (excitation wavelength, 488 nm) in a spectrofluorometer (RF-5301, Shimadzu, Japan). Estimation of Biofilm Biomass by Crystal Violet Assay. To study the antibiofilm activity of GOx-immobilized CS-NPs, the S. aureus-containing biofilm was prepared in a microplate well with an NB medium. After the biofilm was cultured for 24 h incubation, the spent medium was decanted, and a fresh medium with GOx/CS-NP samples (10 μL) was added to the well with biofilm. After overnight incubation, the spent medium was decanted, and the well was washed with sterile water (200 μL) to remove nonadherent bacteria. The well was air-dried for 45 min, and 0.1% (v/v) crystal violet solution (150 μL) was added to each well and incubated for 30 min to stain the biofilm. The crystal violet stain within the biofilm was solubilized with 95% ethanol (200 μL), and the biofilm biomass was estimated by measuring the absorbance of ethanol-solubilized dye solution at 590 nm in a microplate reader (Tecan Trading AG, Männedorf, Switzerland). The biomass obtained for untreated sample was also estimated as a control. Confocal Laser Scanning Microscopy for Biofilm Analysis. S. aureus biofilm was grown on a coverslip by following a standard methodology.44 The 1 day-grown biofilm was treated with 40 μL of GOx-immobilized CS-NPs for 24 h. Then, the media were removed, and the wells were washed twice with sterile Milli-Q water (200 μL) to remove nonadherent bacteria. Biofilm samples were fluorescently labeled with cFDA-SE at a working concentration of 50 μM in PBS.45 After removing excess and unbound cFDA-SE molecules and airdrying, stained biofilm was observed with confocal laser scanning microscopy (CLSM) (LSM 700, Carl-Zeiss, Germany). Images were recorded at the green channel (excitation 488 nm and emission 518 nm). Z-stack images were acquired using IMARIS software (Bitplane AG, Zurich, Switzerland). Fabrication and Characterization of Biocatalytic Gauzes. GOx-immobilized CS-NPs (0.1 μg of CS-NPs) in 1 mL of PBS were uniformly spread onto the cotton gauzes (diameter: 1 cm). For control sample, only PBS solution was spread in the same condition. Each sample was freeze-dried for 24 h. Zone of inhibition assay was carried out to quantitatively evaluate the antibacterial performance of biocatalytic gauzes. After spreading the S. aureus suspension (106 cfu/ mL) uniformly on the solid NB-agar plates, the fabricated biocatalytic gauzes (circular disks, diameter of 10 mm) were placed on the agar plates and incubated at 37 °C for 24 h. The annular radius of the C


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Biomacromolecules formed inhibition zone around each disk was measured to evaluate the antibacterial effects. The experiments were conducted in triplicate.

explained by the conversion of chitosan amine group from positive to neutral. When the enzymes were immobilized onto CS-NPs via covalently attachment approach, the zeta potential becomes negative, which can be explained by the isoelectric points of each GOx (pI 4.2) and bCA (pI 5.7). The EC samples showed higher zeta potential compared to CA counterparts, indicating the increased enzyme loading upon GA cross-linking. On the other hand, EPC and Mag-EPC preparations of anionic GOx and bCA demonstrated highly positive zeta potentials, implying that precipitation and crosslinking affects both enzyme loading and particle surface charge. It may be hypothesized that ammonium cations from ammonium sulfate precipitation are retained in the EPC matrix, thereby improving the positive surface charge of EPC and Mag-EPC. In both GOx and bCA, the absolute zeta potentials of EPC and Mag-EPC were higher than those of CSNPs, CA, and EC. This may explain the good dispersion of EPC preparations in aqueous solutions because of increased electrostatic repulsion between adjacent EPC particles. These results strongly suggest that enzyme immobilization onto CSNPs via EPC and Mag-EPC approaches effectively prevents CS-NPs aggregation. Dynamic light scattering (DLS) analysis was performed to investigate the particle size distribution of CS-NPs and all enzyme-immobilized CS-NP preparations in aqueous buffer solution (Figure 3). The particle size of CS-NPs was estimated to be 98 ± 28 nm, which was used for the preparation of both GOx- and bCA-immobilized CS-NPs. When GOx was immobilized onto CS-NPs, the particle sizes of CA, EC, EPC, and Mag-EPC samples were 397 ± 110, 256 ± 67, 895 ± 247, and 920 ± 177 nm, respectively. The DLS results of bCA showed a similar tendency of increasing size in order of CA, EC, EPC, and Mag-EPC with average diameters of 157 ± 44, 172 ± 47, 260 ± 70, and 267 ± 63 nm, respectively. Interestingly, GOx-immobilized CS-NPs were larger than bCA immobilization counterparts, reflecting the larger size of GOx (6.0 × 5.2 × 7.7 nm46) than bCA (4.1 × 4.1 × 4.7 nm47). Other intrinsic enzyme properties such as number of lysine residue and surface hydrophobicity showed little difference between GOx and bCA (Table S1), suggesting that the different size of the enzyme molecules is the main factor resulting in the different sizes of enzyme-immobilized CS-NPs, especially for EPC and Mag-EPC preparations. Figures 4 and S3 show the SEM images of CS-NPs, CA, EC, EPC, and Mag-EPC samples prepared by using both GOx and bCA. Interestingly, only the EPC and Mag-EPC samples were spherical in both SEM and cryo-transmission electron

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RESULTS AND DISCUSSION Synthesis and Characterization of GOx and bCAImmobilized CS-NPs. CS-NPs were prepared by ionotropic gelation, and model enzymes of GOx and bCA were subsequently immobilized onto the surface of CS-NPs via CA, EC, EPC, and Mag-EPC methodologies (Figure 1). Particle characteristics of enzyme-immobilized CS-NPs were examined in terms of aqueous dispersion, surface charge, morphology, and size. The dispersion and aggregation of GOxor bCA-immobilized CS-NPs samples were determined after incubation in aqueous solution (100 mM sodium phosphate) overnight under shaking at 200 rpm. CA- and EC-based CSNPs showed serious particle aggregation while EPC- and MagEPC-based CS-NPs maintained good dispersion (Figure 2). In

Figure 2. Observation of (a) GOx- and (b) bCA-immobilized CSNPs in aqueous solution with CS-NPs as a control. The pictures were taken after the samples were shaken (200 rpm) overnight.

particular, CS-NPs without enzyme immobilization resulted in one large clump of aggregated CS-NPs. Both GOx and bCA exhibited similar trends of serious aggregation with CA and EC samples while good dispersion with EPC and Mag-EPC samples. Simultaneously, zeta potential analysis was performed to examine quantitatively the surface charges of the prepared particles (Figure S2). CS-NPs showed slightly positive zeta potentials as a result of the TPP forcing the chitosan amine groups toward the surface and exposed to the medium while the TPP remains inside the CS-NPs. When considering the pH optima for each enzyme (GOx = pH 7.0; bCA = pH 7.6), the reduced net positive charge of CS-NP for bCA can be

Figure 3. Size distribution of (a) GOx- and (b) bCA-immobilized CS-NPs together with CS-NPs from the DLS analysis. D


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Figure 4. SEM images of (a) CS-NPs, (b) GOx-, and (c) bCA-immobilized CS-NPs via four different approaches, which are CA, EC, EPC, and Mag-EPC.

microscopy (TEM) images (Figure S4), suggesting that those samples can maintain their spherical form upon drying because of effective cross-linking of precipitated enzyme molecules. The average sizes of EPC and Mag-EPC were 522 ± 135 and 632 ± 150 nm, respectively, for GOx, while they were 225 ± 45 and 243 ± 53 nm, respectively, for bCA. The larger molecular size of GOx versus bCA resulted in larger sizes of the immobilized preparations. The smaller size of EPC and Mag-EPC than those from DLS analysis reveals the shrinkage of particle volume upon drying for SEM imaging. Both DLS and SEM analyses clearly show that the particle size of MagEPC was larger than that of EPC in both solution and dried conditions. The larger size of Mag-EPC than EPC can be explained by the coimmobilization of magnetic nanoparticles (50 nm diameter). The TEM image of Mag-EPC-GOx shows the presence of magnetic nanoparticles in conjunction with cross-linked enzymes, likely a result of the cross-linking of amine-functionalized magnetic nanoparticles with GOx on the surface of CS-NPs (Figure S5). Enzyme Activities and Stabilities of GOx- and bCAImmobilized CS-NPs. In the case of GOx-immobilized CSNPs, the activity of Mag-EPC was approximately 26-fold higher than that of the CA formulation and 12-fold higher than the EC formulation (Figure 5a). The Mag-EPC was approximately 13% more active than the EPC formulation. We also checked the enzyme activity of bCA-immobilized CSNPs (Figure S6a). Similar to GOx, the EPC protocol including magnetic particle incorporation demonstrated largely increased activities compared to CA and EC formulations; in the case of Mag-EPC-bCA, the preparation was approximately 22-fold more active than the CA preparation and 2.9-fold more active than the EC preparation. The higher activity for the two EPC preparations versus the CA and EC preparations is likely due to the higher enzyme loading achieved as a result of efficient cross-linking of precipitated enzyme molecules under close contact. It should be noted that iron oxide nanoparticles are known to possess peroxidase-like activity, particularly under acidic conditions,48 and therefore could impact the measurement of GOx activity using HRP; such activity is dramatically lower under neutral pH. To test this, we measured the peroxidase activity of our Mag-EPC-GOx preparations at pH 7 and found that the activity at pH 7 was approximately 120-fold lower than at pH 3.5 (data not shown). Thus, the magnetic nanoparticle does not interfere with the Mag-EPC-GOx activity determination.

Figure 5. (a) Activities of GOx-immobilized CS-NPs. One unit of GOx activity is defined by the enzyme amount that catalyzes the oxidation of 1 μmol of glucose to D-gluconolactone and H2O2 per 1 min in 100 mM sodium PB (pH 7.0). (b) Stability of GOximmobilized CS-NPs at room temperature.

The stabilities of the four preparations were determined in aqueous buffer (100 mM sodium phosphate, pH 7.0) at room temperature. As shown in Figure 5b, CA, EC, EPC, and MagEPC retained 25, 49, 83, and 86% of their initial activities up to 16 days of incubation, respectively. The inactivation rate constants of CA, EC, EPC, and Mag-EPC, determined from the slope of first-order enzyme inactivation plots (Figure S7), were 0.091, 0.046, 0.008, and 0.013 day−1, respectively. Higher stabilities of EC, EPC, and Mag-EPC compared with CA suggest that multipoint covalent linkages between enzyme molecules upon GA treatment prevent enzyme denaturation.37 Furthermore, EPC and Mag-EPC were more stable than EC, indicating that the enzyme precipitation step is critical in improving the enzyme stability, perhaps by reducing the distance between enzyme molecules. In other words, tightly cross-linked enzyme aggregates may have greater resistance to E


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Biomacromolecules denaturation, resulting in increased stability of EPC and MagEPC. Mag-EPC can be captured by using a magnet within 30 s (Figure S8), which provides a facile option of magnetic separation for its recycled uses. We also performed enzyme leaching studies for the four immobilized GOx preparations by incubating the immobilized enzymes for 5 days and then isolating the incubation supernatants (Figure S9). In all cases, the GOx activity in the supernatant was less than 5% of the immobilized enzyme preparations. In the case of bCA-immobilized CS-NPs, EC, EPC, and Mag-EPC retained 35, 58, and 51% of their initial activities after 16 days of incubation, respectively, while CA showed only 6.0% of its initial activity even after incubation in the same condition for 5 days (Figure S6b). The thermal stabilities of bCA-immobilized CS-NPs were also measured at 50 °C (Figure S6c). Thermal stabilities of bCA samples showed similar trends as that at room temperature. These results support that our strategy for enzyme immobilization could be extended to other enzymes. Antimicrobial Applications of GOx-Immobilized CSNPs. The antimicrobial activity of GOx is a result of H2O2 generation. To assess the antimicrobial activity of GOx, we used the four GOx/CS-NP preparations and followed the growth inhibition of S. aureus in suspension cultures using a simple turbidity assay. As shown in Figure 6, all four GOx/CS-

Figure 7. cfu assay against S. aureus treated with GOx-immobilized CS-NPs, which are CA, EC, EPC, and Mag-EPC. In control sample, only PBS solution was treated. (a) Time-kill assay at various time intervals and (b) cfus following 3 h incubation.

killing with the other two controls, one being Mag-EPC (without GOx) and the second being Mag-EPC-BSA, the latter containing the inert BSA bound to the magnetic nanoparticles. As shown in Figure S10, no inhibition of S. aureus growth was obtained with these control materials in actively growing suspensions cultures. The MBCs of EPC-GOx and Mag-EPCGOx were 30 and 20 ng CS-NPs/mL, respectively (Figure S11). These results strongly indicate that high antibacterial activities of EPC-GOx and Mag-GOx were attributed to the high enzyme loadings as a result of efficient cross-linking of precipitated enzyme molecules. We confirmed that cell killing was a result of H2O2 by using the cFDA-SE leakage assay to quantify damage to the bacterial cell membrane.44 The EPC and Mag-EPC showed higher levels of fluorescent dye leakage than CA and EC samples (Figure 8), which is consistent with the observed antibacterial activities due to the GOx/CS-NP preparations. Bacteria in biofilms are inherently more difficult to be killed than in suspension cultures because of the protection by selfproduced exopolymer matrices.49 To assess whether GOx/CSNP preparations were effective against S. aureus-containing biofilms, we assessed cell killing using several assays. First, 3D CLSM projection of fluorescently stained biofilms clearly showed that the addition of GOx-immobilized CS-NPs results in reduced confluence of bacteria present within the biofilm matrix compared to the no-enzyme control (Figure 9), and this was particularly evident for EPC and Mag-EPC preparations. Crystal violet straining assay was also conducted to estimate quantitatively biofilm formation (Figure 10).45 The EPC and Mag-EPC preparations showed the highest antibacterial activity against the S. aureus-containing biofilm, consistent with the results with S. aureus suspension cultures.

Figure 6. Growth curves of S. aureus treated with GOx-immobilized CS-NPs, which are CA, EC, EPC, and Mag-EPC.

NP preparations inhibited microbial growth when compared to control sets (no treatment). Antimicrobial activity was strikingly high for the EPC and Mag-EPC preparations containing GOx. As previously shown in zeta potential analysis result, the positive surface charge of EPC and Mag-EPC can contribute to their favorable interaction with marginally negative charged surface of S. aureus. Nevertheless, such potent growth inhibition is presumably due to high enzyme loadings per carrier particle and good dispersion in aqueous growth medium, which accelerate H2O2 generation and facilitate efficient H2O2 mass transfer needed for bactericidal activity. In colony forming assay, lower cfu represents higher antibacterial activity. As shown in Figure 7, 100% bacterial killing against S. aureus was observed in EPC-GOx (50 ng of CS-NPs/mL) and Mag-EPC-GOx (50 ng of CS-NPs/mL) after the incubation at 37 °C for 3 h. Unlike EPC-GOx and Mag-EPC, there is no significant bacterial killing with control (no GOx), CA-GOx, and EC-GOx. There is also no bacterial F


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Figure 10. Quantification of S. aureus biofilm after treatment with GOx-immobilized CS-NPs prepared via CA, EC, EPC, and Mag-EPC. Amount of S. aureus biofilm was estimated via crystal violet absorbance at 590 nm.

Figure 8. cFDA-SE dye leakage assay in S. aureus cells treated with GOx-immobilized CS-NPs, which are CA, EC, EPC, and Mag-EPC.

Furthermore, the Mag-EPC (without GOx) and Mag-EPCBSA controls showed no antimicrobial activity against the biofilm culture (Figure S12). The GOx-immobilized CS-NPs were applied for the fabrication of biocatalytic gauzes. There was no observed inhibition zone for bare gauze (control) against S. aureus, as shown in Figure 11a. In contrast, the inhibition zone was clearly observed for the gauzes with GOx-immobilized CSNPs. The average annular radius of the inhibition zone for EPC-GOx gauze is 6.1 ± 0.3 mm, compared to 3.0 ± 0.5 and 4.0 ± 0.4 mm for CA- and EC-GOx gauze against S. aureus, respectively (Figure 11b−d), indicating that biocatalytic gauze with EPC-GOx has excellent antibacterial activity because of the high enzyme loading via EPC approach. It has a great potential for applications as antibacterial biocatalytic gauze for medical dressing.

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CONCLUSIONS We have demonstrated the successful immobilization and stabilization of GOx and bCA on CS-NPs via an EPC methodology. EPC not only improved enzyme loading and stability but also maintained good dispersion of the resulting GOx-immobilized CS-NPs. Mag-EPC also adds value to the EPC approach by enabling facile magnetic separation. With the aid of both colloidal stability and high enzyme loading, EPCand Mag-EPC-GOx could be used to effectively inhibit the proliferation of S. aureus in suspension and biofilm cultures.

Figure 11. Zone of inhibition assay against S. aureus for the gauzes with GOx-immobilized CS-NPs, which are CA, EC, EPC, and MagEPC. In control sample, only PBS solution was spread onto bare gauze.

These results also suggest that EPC preparations of CS-NPs can be used for generating highly dispersed and homogeneous nanobiocatalysts based on the highly biocompatible natural

Figure 9. Confocal microscopy analysis of S. aureus biofilm after treatment with GOx-immobilized CS-NPs. (a) Control, (b) CA, (c) EC, (d) EPC, and (e) Mag-EPC. Scale bar represents 100 μm. G


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Biomacromolecules

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polymer chitosan. This opens up potential applications in both environmental remediation and biomedical fields.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.9b00152. Intrinsic properties of GOx and bCA, schematic representation of CS-NP preparation, zeta potentials of enzyme-immobilized CS-NPs, SEM images of CS-NPs and GOx-immobilized CS-NPs, cryogenic TEM images of EPC-GOx and Mag-EPC-GOx, TEM image of MagEPC-GOx, activities and stabilities of bCA-immobilized CS-NPs, time courses of relative activities of GOximmobilized CS-NPs, magnetic separation of Mag-EPCGOx, comparison of activities between leached GOx and immobilized GOx, and cfu assay treated with two controls; Mag-EPC (without GOx) and Mag-EPC-BSA, determination of MBCs of EPC-GOx and Mag-EPCGOx, and quantification of S. aureus biofilm after treatment with two controls; and Mag-EPC (without GOx) and Mag-EPC-BSA (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (J.S.D.). *E-mail: [email protected] (J.K.). ORCID

Seok-Joon Kwon: 0000-0002-5466-1993 Moon Il Kim: 0000-0003-1844-0939 Jonathan S. Dordick: 0000-0001-7802-3702 Jungbae Kim: 0000-0001-8280-7008 Author Contributions

K-M.Y., J.Y., and M.D.A. contributed equally. The manuscript was written through contributions from all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Global Research Laboratory Program (2014K1A1A2043032) and the Nano Material Technology Development Program (2014M3A7B4052193) through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT. This work was also supported by the Korea Institute of Energy Technology Evaluation and Planning (KETEP) and the Ministry of Trade, Industry & Energy (MOTIE) of the Republic of Korea (no. 20182010600430).

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ABBREVIATIONS GOx, glucose oxidase; bCA, bovine carbonic anhydrase; CSNPs, chitosan nanoparticles; CA, covalent attachment; EC, enzyme coating; EPC, enzyme precipitate coating; Mag-EPC, magnetic nanoparticle-incorporated EPC

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REFERENCES

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Enzyme-Immobilized Chitosan Nanoparticles as Environmentally

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