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Surface Functionalized Electrospun Biodegradable Nanofibers for Immobilization of Bioactive Molecules

Taek Gyoung Kim and Tae Gwan Park*
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, South Korea

A blend mixture of biodegradable poly( -caprolactone) (PCL) and poly(D,L-lactic-co-glycolic acid)-poly(ethylene glycol)-NH2 (PLGA-b-PEG-NH2) block copolymer was electrospun to produce surface functionalized nanofibers. The resulting nanofibrous mesh with primary amine groups on the surface was applied for immobilization of biologically active molecules using lysozyme as a model enzyme. Lysozyme was immobilized via covalent conjugation by using a homobifunctional coupling agent. The nanofibrous mesh could immobilize a far greater amount of lysozyme on the surface with concomitantly increased activity, primarily due to its larger surface area, compared to that of the solvent casting film. It was also found that the enzyme immobilization process slightly altered thermal and pH-dependent catalytic activity profiles compared to those of native lysozyme. The results demonstrated the surface functionalized electrospun nanofibrous mesh could be used as a promising material for immobilizing a wide range of bioactive molecules.

Introduction
Surface immobilized nanostructures with bioactive molecules have provided great opportunities for developing diagnostic sensors, bioprobes, and biomedical devices (1-4). The nanosized materials could present high surface areas, onto which many bioactive molecules could be immobilized in high density. This ultimately led to the miniaturization of the biological devices with high sensitivity (5, 6). A wide range of biomolecules such as enzymes, antibodies, peptides, and DNA have been immobilized onto a large number of nanomaterials including inorganic nanoparticles, nanotubes, organic colloids, and various fibrous materials. Significant improvements in efficiency and sensitivity of immobilized molecules for recognizing their counterparts could be achieved (7-11). Recently, electrospun polymeric nanofibers have been used for biomedical applications as a result of their unique nanoscale structures (12-14). A wide array of nanofibers with a diameter ranging from submicron to a few microns shows extraordinary properties such as dramatically increased surface/volume ratio, excellent mechanical strength, and highly open porous structure. Thus, the nanofibers have been explored as an extracellular matrix (ECM) mimicking tissue engineering scaffolds, novel carriers for bioactive drugs, and filtrations for biomolecules (15-20). Although several techniques are available to produce various types of nanofibers such as self-organization, phase separation, the template method, and electrospinning (21-23), electrospinning is the most cost-effective and straightforward method to generate continuous nonwoven nanofibers. We recently demonstrated immobilization of cell adhesive Gly-ArgGly-Asp-Tyr (GRGDY) peptide on the surface of electrospun poly(D,L-lactic-co-glycolic acid) (PLGA) nanofibrous mesh (23). A blend mixture of PLGA and PLGA-b-PEG-NH2 diblock copolymer was electrospun to produce nanofiber meshes having primary amino groups on their surface, and then the amine functionalized meshes were covalently modified with RGD
* To whom correspondence should be addressed. Tel: +82-42-869-2621. Fax: +82-42-869-2610. E-mail: tgpark@kaist.ac.kr.
10.1021/bp060039t CCC: $33.50

peptides. The RGD immobilized nanofibrous mesh showed improvement of cellular attachment, spreading, and proliferation. In this study, the surface of biodegradable nanofibrous mesh was functionalized with primary amine groups and used for immobilization of enzymes. High surface-to-volume ratio of the nanofibrous mesh provides definite advantages for the enzyme immobilization, as compared to any other nonporous materials. Biocompatible and biodegradable electrospun nanofibers immobilized with bioactive molecules could be potentially used for implantable devices or wound dressing materials. Lysozyme as a model biologically functional molecule was immobilized on the surface of the nanofibers via covalent conjugation. Biodegradable polymeric nanofibers composed of polycaprolactone and PLGA-b-PEG-NH2 diblock copolymer were produced by electrospinning. The surface aminated biodegradable nanofibers were used for the covalent immobilization of lysozyme using amine-reactive coupling agents. Lysozymeimmobilized electrospun nanofibrous mesh was thoroughly characterized, as compared to lysozyme-immobilized solvent casting nonporous films.

Materials and Methods

Materials. PLGA (lactic:glycolic acid ratio 50:50, RG504H, MW 45,000) was purchased from Boehringer Ingelheim (Ingelheim, Germany). Poly( -caprolactone) (PCL, MW 65,000) was supplied by Aldrich (Milwaukee, WI). Lysozyme (from chicken egg white, 50,000 U/mg protein) (E.C. 3.2.1.17, mucopeptide N-acetylmuramylhydrolase), Micrococcus lysodeikticus (dried cells), polyoxyethylene-bis(amine) (PEG-diamine, MW 3,350), fluorescamine, ninhydrin reagent, N-hydroxysuccinimide (NHS), and dicyclohexylcarbodiimide (DCC) were obtained from Sigma (St. Louis, USA). Ethyleneglycol-bis(succinimidylsuccinate) (EGS) was from Pierce (Rockford, IL). Preparation of Amine Terminated PEG-b-PLGA Diblock Copolymers. PLGA-b-PEG-NH2 was prepared using RG504H (PLGA) according to a method previously reported (24). Briefly, a carboxylic acid of the PLGA terminal end group was activated with DCC and NHS and then conjugated a primary amino group

2006 American Chemical Society and American Institute of Chemical Engineers Published on Web 06/24/2006

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Figure 1. Schematic of electrospinning setup device.

of PEG diamine. An excess amount of PEG diamine (10 times molar excess) was used to prevent the formation of PLGAPEG-PLGA triblock copolymers. The coupling reaction was performed in methylene chloride under magnetic stirring for 12 h at room temperature. PLGA-b-PEG-NH2 copolymer was precipitated by slowly dropping into cold ethanol. The collected gel mass was washed with an excess amount of ethanol and dried under vacuum. The synthesis of the diblock copolymers was confirmed by 1H NMR and differential scanning calorimetry. Fabrication of Aminated PCL/PLGA Blend Nanofibers via Electrospinning. The electrospinning apparatus as shown in Figure 1 was used to fabricate nanofibrous mesh. A blend mixture of PCL and PLGA-b-PEG-NH2 was dissolved in a mixed solvent of N,N-dimethyl formamide (DMF) and tetrahydrofuran (THF) (1/1 v/v). Different blend ratios of PCL/PLGAb-PEG-NH2 at 90/10, 70/30, and 50/50 (w/w) were electrospun at a fixed concentration (20% w/v). For electrospinning, each polymer solution was added into a syringe with a 24 gauge stainless steel needle. The polymer solution was delivered to the needle by a syringe pump (model 210, KD Scientific Inc., USA) at a constant feed rate of 20 L/min and connected to a high voltage DC power supplier (CPS-40 K03VIT, Chungpa EMT Co., Korea). The electrospinning was performed in an ambient condition and at a voltage range from 14 to 16 kV. Randomly oriented fibrous mesh was collected on a grounded drum rotating at 500 mm/s. The resulting mesh was placed in a vacuum oven for 2 days to remove residual solvent. For a control study, solvent casting films were prepared using the same blend ratios of PCL/PLGA-b-PEG-NH2. The polymer was first dissolved at a concentration of 4 wt % in chloroform. The resulting solution was then cast onto a glass Petri dish (diameter 50 mm) with Teflon coating. The samples were placed under laminar flow for 24 h to allow the solvent to evaporate and then were vacuum-dried. Characteristics of Electrospun Nanofibrous Mesh. The morphology of the electrospun nanofibers was examined with scanning electron microscopy (SEM, Philips 535M, Netherlands) after gold coating. From the SEM images, the fiber diameter was determined by using an image analyzer (Image J, developed by the U.S. National Institutes of Health). The electrospun meshes were punched into circular pieces with a diameter of 20 mm, followed by measurement of the thickness by using a

micrometer. The apparent density (Fa) and porosity ( ) were obtained from the following equations:

Fa ) ) 1-

( )

m r2h

Fa 100 Fb

where m, r, and h are mass, radius, and thickness of the circular piece of electrospun mesh. Bulk densities (Fb) of PCL and blend polymer were 1.15 and 1.21 g/cm3, respectively. Analysis of Surface Amino Groups. The amounts of primary amino groups on the surface of casting films and electrospun meshes were determined by using a coupling reaction of an amine reactive fluorescamine dye with primary amino groups. Briefly, 10 mg of sample was prewetted with 70% ethanol and washed with deionized water successively. To fully expose the hydrophilic block of -PEG-NH2 in the diblock copolymer on the surface, the sample was hydrated in phosphate buffered saline (PBS, pH 7.4) solution for 24 h at room temperature. Subsequently, the sample was soaked in 3 mL of 50 mM borate buffer (pH 9) containing 300 L of fluorescamine solution (0.3 mg/mL in acetone) for 5 min with vortexing and 15 min with gentle shaking. The resulting yellowish films or meshes were washed with methanol and observed by using a laser scanning confocal microscope (LSCM, Carl Zeiss LSM5100, Germany). To quantify the surface amine concentration, the fluorescamineconjugated samples were dissolved in 1 mL of tetrahydrofuran (THF). Fluorescence intensity of the polymer solution was measured by a spectrofluorophotometer (RF-5391PC, Shimadzu, Japan) at 390 nm of excitation wavelength and 475 nm of emission wavelength. The amount of surface amino group was then calculated from a calibration curve that was constructed by using PEG diamine and PCL dissolved in THF. Immobilization of Lysozyme. For lysozyme immobilization, aminated PCL/PLGA films or meshes with varying surface amine amounts were prewetted and hydrated as described above. Each sample (10 mg) was immersed in 1.5 mL of PBS containing 3 mol of ethylene glycol-bis(sulfosuccinimidylsuccinate) (EGS) with gentle agitation for 1 h at room temperature. After being rinsed with PBS three times, the activated sample was put into 3 mL of PBS containing 1.5 mol of lysozyme (21.5 mg) under shaking for 24 h at 4 C. At the end of the coupling reaction, the lysozyme-immobilized films or meshes were rinsed with PBS and then were lyophilized.

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Table 1. Characteristics of Electrospun Nanofibers with Varying Polymer Compositions
PCL:PLGA-b-PEG-NH2 100:0 fiber diameter (nm) apparent density (g/cm3) porosity (%) 459 ( 289 0.21 ( 0.05 82 ( 4 90:10 406 ( 195 0.25 ( 0.02 79 ( 2 70:30 386 ( 128 0.28 ( 0.08 77 ( 6 50:50 352 ( 168 0.30 ( 0.05 75 ( 4

Analysis of surface Immobilized Lysozyme. Fourier transformed infrared spectroscopy (FTIR spectrometer, Bruker Equinox 55, Germany) was used for identifying the immobilized lysozyme on the surface of electrospun mesh. The amount of immobilized lysozyme on the surface was determined using a colorimetric ninhydrin method. The lysozyme-immobilized films or meshes were put into 2 mL of 6 N HCl, autoclaved at 120 C for 15 min, and then cooled for 1 h at room temperature. The resultant solution was neutralized with 1.5 mL of 5 N NaOH and 7.5 mL of 1 M sodium acetate buffer (pH 4.7). One milliliter of ninhydrin reagent solution was added to the aliquot (2 mL) from the solution, followed by heating at 100 C for 5 min, to ensure complete color development. After the purple colored solution cooled to room temperature, 5 mL of 95% ethanol was added. The amount of lysozyme was determined by using UV spectrophotometer (UV-1601, Shimadzu, Japan) at 570 nm. A standard calibration curve was constructed by using the aminated PCL/PLGA films and a known amount of lysozyme. Measurement of Lysozyme Activity. The catalytic activity of immobilized lysozyme was measured, in triplicate, by the method of M. lysodeikticus cell lysis. After being prewetted, 20 mg of lysozyme-immobilized films or meshes was added to 5 mL of 0.015% (w/v) M. lysodeikticus suspension in 66 mM potassium phosphate buffer (pH 6.24), followed by shaking the reaction mixture for 10 min, and the decrease in absorbance at 450 nm was measured using a UV spectrophotometer. Serially diluted free lysozyme was used to construct a calibration curve. Effect of Temperature and pH on the Activity of Lysozyme. Lysozyme activity was measured over the temperature range of 13-52 C and pH range of 4-10 to evaluate the effect of temperature and pH on the activity for free lysozyme and lysozyme-immobilized cast films and meshes. For determining temperature and pH effect, 400 U/mL of free lysozyme and 20 mg of lysozyme-immobilized film or meshes were incubated in potassium phosphate buffer (pH 6.24) at each temperature point for 1 h or at 25 C for measuring pH effect, followed by addition to 5 mL of M. lysodeikticus suspension, which was incubated at the same temperature or prepared by buffers with each pH point. The lysozyme activity was normalized to the highest value.

Results and Discussion

Surface Amine Functionalized Nanofibers. The morphology of electrospun nanofibrous meshes can be influenced by various parameters including polymer solution property (surface tension, conductivity, and viscosity), spinning voltage, flow rate, motion of collector, and distance between needle tip and collector (25-27). Generally, electric potential and polymer solution viscosity are important factors in determining the morphological structure of nanofibrous mesh. To obtain suitable electrospun nanofibers with more amine functionalized surface and stable nanofibrous structure, a mixture of PCL and PLGAb-PEG-NH2 with different blend ratios was electrospun. Defectfree and stable nanofibers could be obtained by varying the blend ratio of PLGA-b-PEG-NH2 to PCL up to 50%, whereas beadand spindle-like structures were produced above a 50% blend ratio. As shown in Table 1, significant change in fiber diameter occurred upon varying the blend ratio between PCL and PLGAb-PEG-NH2. The average diameter of electrospun nanofibers decreased from 459 to 352 nm as the blend ratio of PLGA-bPEG-NH2 to PCL increased. This was due to the decreased viscosity of the electrospinning polymer solution with increase in the amount of the more hydrophilic PLGA-b-PEG-NH2 in the blend mixture (23). Density and porosity of the resultant

electrospun mesh were largely affected by the diameter of nanofibers. The nanofibers with a smaller diameter apparently reduced free pore volume in the mesh, resulting in a highly packed fibrous structure. Figure 2 A and B exhibit the morphological difference between pure PCL and 50% blend nanofibrous meshes. More branched and interconnected structures can be seen for the 50% blend mesh. This was also primarily caused by the effect of polymer solution viscosity. Previous study revealed that the fibers had an irregular and split morphology with numerous junctions at the low solution viscosity, whereas they had a regular and cylindrical morphology with a uniform diameter at the high solution viscosity (28). When the blend ratio of PLGA-b-PEG-NH2/PCL varied from 10% to 50%, the measured surface amine density of the meshes greatly increased from 2.1 to 15.2 nmol/cm2, whereas those of the films increased slightly from 1.01 to 3.42 nmol/cm2, as shown in Figure 3. This is most likely due to the combined effect of increasing blend ratio of PLGA-b-PEG-NH2 and a nanoscale fiber diameter. When the blending ratio of diblock copolymer increased, overall surface amine density of both electrospun mesh and casting film increased concomitantly. For the electrospun nanofibers, however, tremendously enlarged surface area significantly contributed to the surface amine density, because amphiphilic PLGA-b-PEG-NH2 would have a greater opportunity to be exposed onto the outer surface. In contrast, for the blend films, the surface amine density increased modestly with the blend ratio as a result of the limited surface area. Figure 2D shows a LCSM image of the aminated nanofibers stained with an amine-reactive dye, fluorescamine. The fluorescence can be observed along with the nanofibers. Thus, the confocal image can prove the presence of amino groups on the surface with a homogeneous distribution. Lysozyme Immobilization and Catalytic Activity. The amine functionalized films and electrospun meshes were conjugated with lysozyme by using a homobifunctional crosslinking reagent, EGS containing two active NHS esters (Figure 4). EGS first reacted with an exposed terminal amino group of PEG to form a stable amide bond, which was subsequently conjugated with an accessible R-amine group present on the N-terminal or an -amine group of lysine in lysozyme to yield the immobilized enzyme. To clear the concern of nonspecific adsorption of lysozyme onto hydrophobic surface of film or mesh, an extensive washing step was added prior to measuring surface lysozyme density. It was likely that nonspecific lysozyme binding onto the blend nanofiber surface was minimal, because lysozyme has a net positive charge at neutral pH, similar to that of an aminated nanofiber surface (electrostatic repulsion). In addition, brush-like PEG chains immobilized on the surface effectively reduced the lysozyme adsorption by steric repulsive action. To investigate the existence of lysozyme on the nanofibers, FT-IR spectra of both as-spun and lysozymeimmobilized nanofibers were measured as shown in Figure 5. It can be seen that compared with the as-spun nanofibers, the lysozyme-immobilized nanofibers show new absorption bands at 1530 (amide II), 1650 (amide I), and 3300 cm-1. In general, the amide I and amide II bands appear as a result of the

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Figure 2. SEM micrographs of (A) PCL nanofibers, (B) PLGA-PEG-NH2/PCL 50% blend nanofibers; (C) higher magnification image and (D) LCSM image of PLGA-PEG-NH2/PCL 50% blend nanofibers stained with fluorescamine.

Figure 3. Surface amine densities of solvent casting films and electrospun nanofibers with varying blend ratios.

stretching vibration of the CdO bonds and a combination of C-N stretching and N-H bending vibrations in the backbone of the protein, respectively. It can be supposed that the broad absorption band at 3300 cm-1 arises from the superposition of stretching vibration of O-H and N-H. The results suggest that lysozyme was successfully immobilized on the surface of nanofibers. The amount of immobilized lysozyme was studied as a function of blend content of aminated PLGA. As demonstrated in Figure 6, the electrospun mesh shows higher amount of immobilized lysozyme as compared with those of the films. Large surface area offers improved lysozyme immobilization efficiency by the presence of more exposed reactive amino groups onto the surface. However, the density of amine groups on the nanofibers was not directly proportional to the amount of immobilized lysozyme; the highest conjugation yield (58%) was at a 30% blend ratio. It is assumed that high packing of

Figure 4. Schematic diagram of lysozyme immobilization process.

nanofibers adversely affected the immobilization efficiency because of diffusional limitation. The cell lysis activities of immobilized lysozyme on the meshes and films with varying blend ratios were shown in Figure 7. The casting film with a 50% blend ratio shows much reduced enzyme activity compared with that of the mesh with the same ratio. It was very likely that the increased surface area of nanofibrous structure in the mesh provided greater chance for the immobilized lysozyme to contact and turn over the substrate. However, it should be noted that the micron-sized substrate (M. lysodeikticus) could not deeply penetrate the submicron sized pores of the mesh. The immobilized lysozyme

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Figure 5. FT-IR spectra of (A) PLGA-PEG-NH2/PCL 50% blend nanofibers and (B) lysozyme-immobilized nanofibers.

Figure 6. Amount of lysozyme immobilized on the surface of films and nanofibers.

Evaluation of Thermal Stability and Optimal pH. To estimate the thermal stability, catalytic activities of immobilized lysozyme on the cast film and the mesh were measured as a function of temperature (Figure 8 A). The immobilized enzymes on both the cast film and the mesh showed an optimal temperature about 45 C, whereas free enzyme had an optimal temperature about 37 C. This result can be explained by restriction of conformational mobility for the immobilized enzyme as a result of covalent anchoring, requiring higher activation energy to react with the substrate. In early literature, activation energies were calculated to be 9.1 and 12.9 kJ/mol for free and immobilized lysozyme, respectively (29). Quite often, the immobilization of an enzyme to a solid surface leads to increase in thermal stability at high temperatures (30). The pH-dependent activities for immobilized lysozyme on the mesh and the cast film were evaluated (Figure 8 B). Free amino groups of lysozyme reacted with -PEG-NH2 by NHS ester to form a neutral amide bond, resulting in reduction of positive charge of the enzyme. Moreover, the charge of the support materials can have influence on the enzyme activity profile in various charged milieu. Thus, it can be expected that the enzyme immobilization process leads to a shift in the value of the optimal reaction pH. Free lysozyme exhibited two maximum peaks at pH 6 and pH 9 (31, 32). Immobilized lysozymes on the cast film and the mesh showed similar pH-dependent activity profiles.

Conclusion
This work describes the surface functionalization of electrospun polymeric nanofibers by bioactive molecules. We successfully fabricated surface amine functionalized nanofibers by electrospinning a polymer blend solution composed of PCL polymer and PLGA-b-PEG-NH2 diblock copolymer. The resultant nanofibrous mesh exhibited a nonwoven nanofibrous structure with micron- or submicron-sized pores and homogeneous surface amino group distribution. When lysozyme was immobilized on the surface, the electrospun mesh exhibited catalytic activities superior to those of the cast film mainly as a result of the enlarged surface area. The results suggest that electrospun nanofibers can be good candidates for surface

Figure 7. Activities of lysozyme immobilized onto films and nanofibers as a function of blend ratio.

inside the pores might not be accessible to the substrate, resulting in reduction of the enzymatic reaction by mass transfer limitation. Nevertheless, it seemed that the effect of large surface area in the mesh overwhelmed the mass transfer effect.

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Figure 8. Effect of (A) temperature and (B) pH on the activity of free and immobilized lysozyme on the nanofiber and film.

immobilization of various bioactive agents for the purpose of catalytic reaction and molecular sensing.

Acknowledgment
This study was supported by a grant (KRF-2004-005-D00070) from the Korea Research Foundation, Korea and the Polymer Technology Institute, Sungkyunkwan University, Korea.

References and Notes

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