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Surface Functionalized Electrospun Biodegradable Nanofibers for Immobilization of Bioactive Molecules

Surface Functionalized Electrospun Biodegradable Nanofibers for Immobilization of Bioactive Molecules

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Surface Functionalized Electrospun Biodegradable Nanofibers for Immobilizationof 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
-glycolicacid)-poly(ethylene glycol)-NH
2
(PLGA-
b
-PEG-NH
2
) block copolymer was electrospun toproduce surface functionalized nanofibers. The resulting nanofibrous mesh with primary aminegroups on the surface was applied for immobilization of biologically active molecules usinglysozyme as a model enzyme. Lysozyme was immobilized via covalent conjugation by using ahomobifunctional coupling agent. The nanofibrous mesh could immobilize a far greater amountof lysozyme on the surface with concomitantly increased activity, primarily due to its largersurface area, compared to that of the solvent casting film. It was also found that the enzymeimmobilization process slightly altered thermal and pH-dependent catalytic activity profilescompared to those of native lysozyme. The results demonstrated the surface functionalizedelectrospun nanofibrous mesh could be used as a promising material for immobilizing a widerange of bioactive molecules.
Introduction
Surface immobilized nanostructures with bioactive moleculeshave provided great opportunities for developing diagnosticsensors, bioprobes, and biomedical devices (
1
-
4
). The nano-sized materials could present high surface areas, onto whichmany bioactive molecules could be immobilized in high density.This ultimately led to the miniaturization of the biologicaldevices with high sensitivity (
5
,
). A wide range of biomol-ecules such as enzymes, antibodies, peptides, and DNA havebeen immobilized onto a large number of nanomaterialsincluding inorganic nanoparticles, nanotubes, organic colloids,and various fibrous materials. Significant improvements inefficiency and sensitivity of immobilized molecules for recog-nizing their counterparts could be achieved (
-
11
).Recently, electrospun polymeric nanofibers have been usedfor biomedical applications as a result of their unique nanoscalestructures (
12
-
14
). A wide array of nanofibers with a diameterranging from submicron to a few microns shows extraordinaryproperties such as dramatically increased surface/volume ratio,excellent mechanical strength, and highly open porous structure.Thus, the nanofibers have been explored as an extracellularmatrix (ECM) mimicking tissue engineering scaffolds, novelcarriers for bioactive drugs, and filtrations for biomolecules(
15
-
20
). Although several techniques are available to producevarious types of nanofibers such as self-organization, phaseseparation, the template method, and electrospinning (
21
-
23
),electrospinning is the most cost-effective and straightforwardmethod to generate continuous nonwoven nanofibers. Werecently demonstrated immobilization of cell adhesive Gly-Arg-Gly-Asp-Tyr (GRGDY) peptide on the surface of electrospunpoly(
D
,
L
-lactic-
co
-glycolic acid) (PLGA) nanofibrous mesh (
23
).A blend mixture of PLGA and PLGA-
b
-PEG-NH
2
diblock copolymer was electrospun to produce nanofiber meshes havingprimary amino groups on their surface, and then the aminefunctionalized meshes were covalently modified with RGDpeptides. The RGD immobilized nanofibrous mesh showedimprovement of cellular attachment, spreading, and proliferation.In this study, the surface of biodegradable nanofibrous meshwas functionalized with primary amine groups and used forimmobilization of enzymes. High surface-to-volume ratio of thenanofibrous mesh provides definite advantages for the enzymeimmobilization, as compared to any other nonporous materials.Biocompatible and biodegradable electrospun nanofibers im-mobilized with bioactive molecules could be potentially usedfor implantable devices or wound dressing materials. Lysozymeas a model biologically functional molecule was immobilizedon the surface of the nanofibers via covalent conjugation.Biodegradable polymeric nanofibers composed of polycapro-lactone and PLGA-
b
-PEG-NH
2
diblock copolymer were pro-duced by electrospinning. The surface aminated biodegradablenanofibers were used for the covalent immobilization of lysozyme using amine-reactive coupling agents. Lysozyme-immobilized electrospun nanofibrous mesh was thoroughlycharacterized, as compared to lysozyme-immobilized solventcasting nonporous films.
Materials and Methods
Materials.
PLGA (lactic:glycolic acid ratio 50:50, RG504H,MW 45,000) was purchased from Boehringer Ingelheim (In-gelheim, Germany). Poly(
-caprolactone) (PCL, MW 65,000)was supplied by Aldrich (Milwaukee, WI). Lysozyme (fromchicken egg white, 50,000 U/mg protein) (E.C. 3.2.1.17, muco-peptide
-acetylmuramylhydrolase),
Micrococcus lysodeikticus
(dried cells), polyoxyethylene-bis(amine) (PEG-diamine, MW3,350), fluorescamine, ninhydrin reagent,
-hydroxysuccinimide(NHS), and dicyclohexylcarbodiimide (DCC) were obtainedfrom Sigma (St. Louis, USA). Ethyleneglycol-bis(succinim-idylsuccinate) (EGS) was from Pierce (Rockford, IL).
Preparation of Amine Terminated PEG-
 b
-PLGA DiblockCopolymers.
PLGA-
b
-PEG-NH
2
was prepared using RG504H(PLGA) according to a method previously reported (
24
). Briefly,a carboxylic acid of the PLGA terminal end group was activatedwith DCC and NHS and then conjugated a primary amino group
* To whom correspondence should be addressed. Tel:
+
82-42-869-2621.Fax:
+
82-42-869-2610. E-mail: tgpark@kaist.ac.kr.
1108
Biotechnol. Prog.
2006,
22,
1108
1113
10.1021/bp060039t CCC: $33.50 © 2006 American Chemical Society and American Institute of Chemical EngineersPublished on Web 06/24/2006
 
of PEG diamine. An excess amount of PEG diamine (10 timesmolar excess) was used to prevent the formation of PLGA-PEG-PLGA triblock copolymers. The coupling reaction was per-formed in methylene chloride under magnetic stirring for 12 hat room temperature. PLGA-
b
-PEG-NH
2
copolymer was precip-itated by slowly dropping into cold ethanol. The collected gelmass was washed with an excess amount of ethanol and driedunder vacuum. The synthesis of the diblock copolymers wasconfirmed by
1
H NMR and differential scanning calorimetry.
Fabrication of Aminated PCL/PLGA Blend Nanofibersvia Electrospinning.
The electrospinning apparatus as shownin Figure 1 was used to fabricate nanofibrous mesh. A blendmixture of PCL and PLGA-
b
-PEG-NH
2
was dissolved in amixed solvent of 
,
 N 
-dimethyl formamide (DMF) and tetrahy-drofuran (THF) (1/1 v/v). Different blend ratios of PCL/PLGA-
b
-PEG-NH
2
at 90/10, 70/30, and 50/50 (w/w) were electrospunat a fixed concentration (20% w/v). For electrospinning, eachpolymer solution was added into a syringe with a 24 gaugestainless steel needle. The polymer solution was delivered tothe needle by a syringe pump (model 210, KD Scientific Inc.,USA) at a constant feed rate of 20
µ
L/min and connected to ahigh voltage DC power supplier (CPS-40 K03VIT, ChungpaEMT Co., Korea). The electrospinning was performed in anambient condition and at a voltage range from 14 to 16 kV.Randomly oriented fibrous mesh was collected on a groundeddrum rotating at 500 mm/s. The resulting mesh was placed ina vacuum oven for 2 days to remove residual solvent. For acontrol study, solvent casting films were prepared using the sameblend ratios of PCL/PLGA-
b
-PEG-NH
2
. The polymer was firstdissolved at a concentration of 4 wt % in chloroform. Theresulting solution was then cast onto a glass Petri dish (diameter50 mm) with Teflon coating. The samples were placed underlaminar flow for 24 h to allow the solvent to evaporate andthen were vacuum-dried.
Characteristics of Electrospun Nanofibrous Mesh.
Themorphology of the electrospun nanofibers was examined withscanning electron microscopy (SEM, Philips 535M, Netherlands)after gold coating. From the SEM images, the fiber diameterwas determined by using an image analyzer (Image J, developedby the U.S. National Institutes of Health). The electrospunmeshes were punched into circular pieces with a diameter of 20 mm, followed by measurement of the thickness by using amicrometer. The apparent density (
F
a
) and porosity (
) wereobtained from the following equations:where
m
,
, and
h
are mass, radius, and thickness of the circularpiece of electrospun mesh. Bulk densities (
F
b
) of PCL and blendpolymer were 1.15 and 1.21 g/cm
3
, respectively.
Analysis of Surface Amino Groups.
The amounts of primaryamino groups on the surface of casting films and electrospunmeshes were determined by using a coupling reaction of anamine reactive fluorescamine dye with primary amino groups.Briefly, 10 mg of sample was prewetted with 70% ethanol andwashed with deionized water successively. To fully expose thehydrophilic block of -PEG-NH
2
in the diblock copolymer onthe surface, the sample was hydrated in phosphate bufferedsaline (PBS, pH 7.4) solution for 24 h at room temperature.Subsequently, the sample was soaked in 3 mL of 50 mM boratebuffer (pH 9) containing 300
µ
L of fluorescamine solution (0.3mg/mL in acetone) for 5 min with vortexing and 15 min withgentle shaking. The resulting yellowish films or meshes werewashed with methanol and observed by using a laser scanningconfocal microscope (LSCM, Carl Zeiss LSM5100, Germany).To quantify the surface amine concentration, the fluorescamine-conjugated samples were dissolved in 1 mL of tetrahydrofuran(THF). Fluorescence intensity of the polymer solution wasmeasured 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 wasthen calculated from a calibration curve that was constructedby using PEG diamine and PCL dissolved in THF.
Immobilization of Lysozyme.
For lysozyme immobilization,aminated PCL/PLGA films or meshes with varying surfaceamine amounts were prewetted and hydrated as described above.Each sample (10 mg) was immersed in 1.5 mL of PBScontaining 3
µ
mol of ethylene glycol-bis(sulfosuccinimidylsuc-cinate) (EGS) with gentle agitation for 1 h at room temperature.After being rinsed with PBS three times, the activated samplewas put into 3 mL of PBS containing 1.5
µ
mol of lysozyme(21.5 mg) under shaking for 24 h at 4
°
C. At the end of thecoupling reaction, the lysozyme-immobilized films or mesheswere rinsed with PBS and then were lyophilized.
Figure 1.
Schematic of electrospinning setup device.
F
a
)
m
π 
2
h
)
(
1
-F
a
F
b
)
×
100
Biotechnol. Prog.,
2006, Vol. 22, No. 4
1109
 
Analysis of surface Immobilized Lysozyme.
Fourier trans-formed infrared spectroscopy (FTIR spectrometer, BrukerEquinox 55, Germany) was used for identifying the immobilizedlysozyme on the surface of electrospun mesh. The amount of immobilized lysozyme on the surface was determined using acolorimetric ninhydrin method. The lysozyme-immobilized filmsor 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 NaOHand 7.5 mL of 1 M sodium acetate buffer (pH 4.7). One milliliterof ninhydrin reagent solution was added to the aliquot (2 mL)from the solution, followed by heating at 100
°
C for 5 min, toensure complete color development. After the purple coloredsolution cooled to room temperature, 5 mL of 95% ethanol wasadded. The amount of lysozyme was determined by using UVspectrophotometer (UV-1601, Shimadzu, Japan) at 570 nm. Astandard calibration curve was constructed by using the aminatedPCL/PLGA films and a known amount of lysozyme.
Measurement of Lysozyme Activity.
The catalytic activityof immobilized lysozyme was measured, in triplicate, by themethod of 
M. lysodeikticus
cell lysis. After being prewetted,20 mg of lysozyme-immobilized films or meshes was added to5 mL of 0.015% (w/v)
M. lysodeikticus
suspension in 66 mMpotassium phosphate buffer (pH 6.24), followed by shaking thereaction mixture for 10 min, and the decrease in absorbance at450 nm was measured using a UV spectrophotometer. Seriallydiluted 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 rangeof 13
-
52
°
C and pH range of 4
-
10 to evaluate the effect of temperature and pH on the activity for free lysozyme andlysozyme-immobilized cast films and meshes. For determiningtemperature and pH effect, 400 U/mL of free lysozyme and 20mg of lysozyme-immobilized film or meshes were incubatedin potassium phosphate buffer (pH 6.24) at each temperaturepoint for 1 h or at 25
°
C for measuring pH effect, followed byaddition to 5 mL of 
M. lysodeikticus
suspension, which wasincubated at the same temperature or prepared by buffers witheach pH point. The lysozyme activity was normalized to thehighest value.
Results and Discussion
Surface Amine Functionalized Nanofibers.
The morphol-ogy of electrospun nanofibrous meshes can be influenced byvarious parameters including polymer solution property (surfacetension, conductivity, and viscosity), spinning voltage, flow rate,motion of collector, and distance between needle tip andcollector (
25
-
27 
). Generally, electric potential and polymersolution viscosity are important factors in determining themorphological structure of nanofibrous mesh. To obtain suitableelectrospun nanofibers with more amine functionalized surfaceand stable nanofibrous structure, a mixture of PCL and PLGA-
b
-PEG-NH
2
with different blend ratios was electrospun. Defect-free and stable nanofibers could be obtained by varying the blendratio of PLGA-
b
-PEG-NH
2
to PCL up to 50%, whereas bead-and spindle-like structures were produced above a 50% blendratio. As shown in Table 1, significant change in fiber diameteroccurred upon varying the blend ratio between PCL and PLGA-
b
-PEG-NH
2
. The average diameter of electrospun nanofibersdecreased from 459 to 352 nm as the blend ratio of PLGA-
b
-PEG-NH
2
to PCL increased. This was due to the decreasedviscosity of the electrospinning polymer solution with increasein the amount of the more hydrophilic PLGA-
b
-PEG-NH
2
inthe blend mixture (
23
). Density and porosity of the resultantelectrospun mesh were largely affected by the diameter of nanofibers. The nanofibers with a smaller diameter apparentlyreduced free pore volume in the mesh, resulting in a highlypacked fibrous structure. Figure 2 A and B exhibit themorphological difference between pure PCL and 50% blendnanofibrous meshes. More branched and interconnected struc-tures can be seen for the 50% blend mesh. This was alsoprimarily caused by the effect of polymer solution viscosity.Previous study revealed that the fibers had an irregular and splitmorphology with numerous junctions at the low solutionviscosity, whereas they had a regular and cylindrical morphologywith a uniform diameter at the high solution viscosity (
28
).When the blend ratio of PLGA-
b
-PEG-NH
2
 /PCL varied from10% to 50%, the measured surface amine density of the meshesgreatly increased from 2.1 to 15.2 nmol/cm
2
, whereas those of the films increased slightly from 1.01 to 3.42 nmol/cm
2
, asshown in Figure 3. This is most likely due to the combinedeffect of increasing blend ratio of PLGA-
b
-PEG-NH
2
and ananoscale fiber diameter. When the blending ratio of diblock copolymer increased, overall surface amine density of bothelectrospun mesh and casting film increased concomitantly. Forthe electrospun nanofibers, however, tremendously enlargedsurface area significantly contributed to the surface aminedensity, because amphiphilic PLGA-
b
-PEG-NH
2
would have agreater opportunity to be exposed onto the outer surface. Incontrast, for the blend films, the surface amine density increasedmodestly with the blend ratio as a result of the limited surfacearea. Figure 2D shows a LCSM image of the aminatednanofibers 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 aminogroups on the surface with a homogeneous distribution.
Lysozyme Immobilization and Catalytic Activity.
Theamine functionalized films and electrospun meshes wereconjugated with lysozyme by using a homobifunctional cross-linking reagent, EGS containing two active NHS esters (Figure4). EGS first reacted with an exposed terminal amino group of PEG to form a stable amide bond, which was subsequentlyconjugated with an accessible
R
-amine group present on theN-terminal or an
-amine group of lysine in lysozyme to yieldthe immobilized enzyme. To clear the concern of nonspecificadsorption of lysozyme onto hydrophobic surface of film ormesh, an extensive washing step was added prior to measuringsurface lysozyme density. It was likely that nonspecific lysozymebinding onto the blend nanofiber surface was minimal, becauselysozyme has a net positive charge at neutral pH, similar tothat of an aminated nanofiber surface (electrostatic repulsion).In addition, brush-like PEG chains immobilized on the surfaceeffectively reduced the lysozyme adsorption by steric repulsiveaction. To investigate the existence of lysozyme on thenanofibers, FT-IR spectra of both as-spun and lysozyme-immobilized nanofibers were measured as shown in Figure 5.It can be seen that compared with the as-spun nanofibers, thelysozyme-immobilized nanofibers show new absorption bandsat 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
Table 1. Characteristics of Electrospun Nanofibers with VaryingPolymer Compositions
PCL:PLGA-
b
-PEG-NH
2
100:0 90:10 70:30 50:50fiberdiameter (nm)459
(
289 406
(
195 386
(
128 352
(
168apparentdensity (g/cm
3
)0.21
(
0.05 0.25
(
0.02 0.28
(
0.08 0.30
(
0.05porosity (%) 82
(
4 79
(
2 77
(
6 75
(
4
1110
Biotechnol. Prog.,
2006, Vol. 22, No. 4

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