Dual-functional electrospun poly(2-hydroxyethyl methacrylate)

Dual-functional electrospun poly(2-hydroxyethyl methacrylate)

Bo Zhang,1 Reza Lalani,2 Fang Cheng,3 Qingsheng Liu,1 Lingyun Liu1

1Department of Chemical and Biomolecular Engineering, University of Akron, Akron, Ohio 443252Department of Biology, University of Akron, Akron, Ohio 443253School of Pharmaceutical Science and Technology, Dalian University of Technology, Dalian 116024,

People’s Republic of China

Received 29 March 2011; revised 14 June 2011; accepted 15 June 2011

Published online 1 September 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.33205

Abstract: Poly(2-hydroxyethyl methacrylate) (pHEMA) has

been widely used in many biomedical applications due to its

well-known biocompatibility. For tissue engineering applica-

tions, porous scaffolds that mimic fibrous structures of natural

extracellular matrix and possess high surface-area-to-volume

ratios are highly desirable. So far, a systematic approach to

control diameter and morphology of pHEMA fibers has not

been reported and potential applications of pHEMA fibers have

barely been explored. In this work, pHEMA was synthesized

and processed into fibrous scaffolds using an electrospinning

approach. Fiber diameters from 270 nm to 3.6 lm were

achieved by controlling polymer solution concentration and

electrospinning flow rate. Post-electrospinning thermal treat-

ment significantly improves integrity of the electrospun mem-

branes in water. The pHEMA microfibrous membranes

exhibited water absorption up to 280% (w/w), whereas the

pHEMA hydrogel only absorbed 70% water. Fibrinogen adsorp-

tion experiments demonstrate that the electrospun pHEMA

fibers highly resist nonspecific protein adsorption. Hydroxyl

groups on electrospun pHEMA fibers were further activated for

protein immobilization. A bovine serum albumin (BSA) binding

capacity as high as 120 mg BSA/g membrane was realized at an

intermediate fiber diameter. The pHEMA fibrous scaffolds func-

tionalized with collagen I significantly promoted fibroblast ad-

hesion, spreading, and proliferation. We conclude that the

electrospun pHEMA fibers are dual functional, that is, they

resist nonspecific protein adsorption meanwhile abundant

hydroxyl groups on fibers allow effective conjugation of bio-

molecules in a nonfouling background. High water absorption

and dual functionality of the electrospun pHEMA fibers may

lead to a number of potential applications such as wound

dressings, tissue scaffolds, and affinity membranes. VC 2011

Wiley Periodicals, Inc. J Biomed Mater Res Part A: 99A: 455–466, 2011.

Key Words: poly(2-hydroxyethyl methacrylate), electrospin-

ning, diameter, low fouling, dual functional

How to cite this article: Zhang B, Lalani R, Cheng F, Liu Q, Liu L. 2011. Dual-functional electrospun poly(2-hydroxyethylmethacrylate). J Biomed Mater Res Part A 2011:99A:455–466.

INTRODUCTION

Poly(2-hydroxyethyl methacrylate) (pHEMA), an FDA-approved biocompatible polymer, has been widely utilizedin a variety of biomedical applications such as contact lens,intraocular lens, drug delivery, dental, and orthopedicimplants.1–7 The polymer, rich of hydroxyl groups, is consid-ered as a low fouling material, which resists nonspecificprotein adsorption and cell adhesion. In addition, thehydroxyl functional groups can be further activated for thebioconjugation of proteins, such as those which can modu-late inflammation and enhance wound healing. It has beenreported that pHEMA hydrogel modified with extracellularmatrix (ECM) proteins such as collagen I and osteopontinpromotes endothelial cell adhesion.8,9 Porous pHEMA hydro-gel with tightly controlled pore size significantly promotesvascular in-growth.10

Tissue engineering, a field emerging during the past dec-ades, is intended to repair, restore, or regenerate the dam-

aged tissues. The generic concept of tissue engineering is tocombine biocompatible scaffolds, human cells, and cell re-sponsive biomolecules to regenerate biological tissues.11,12

Scaffolds that mimic fibrous structures of natural ECM aredesirable for tissue engineering applications.12,13 Electro-spinning is a unique method capable of producing continu-ous fibers with diameters ranging from several tens of nano-meters to a few microns, a size range otherwise difficult toachieve with conventional fiber fabrication techniques.13–15

The process, in its simplest form, consists of a positivelycharged capillary filled with a polymer solution, a groundedcollector, and a dc high voltage supply. Once the electrostaticforce is greater than the surface tension of the polymer solu-tion at the capillary tip, a polymer jet is created, which thentravels from the capillary tip to the grounded target andforms continuous polymer fibers. Electrospun fibers mimicthe structures of the natural ECM morphologically and pos-sess large surface-area-to-volume ratio and high porosity

Correspondence to: L. Liu; e-mail: [email protected]

Contract grant sponsor: Cleveland Clinic Foundation/Clinical Tissue Engineering Center; contract grant number: TECH-09-006A

Contract grant sponsor: University of Akron Faculty Research Grant

VC 2011 WILEY PERIODICALS, INC. 455

with interconnected pores,13,16–17 making them promisingcandidates as tissue scaffolds. The method is simple and ver-satile, able to process a wide range of materials.17 Manypolymers, for example, poly(e-caprolactone) (PCL), poly(lac-tic acid) (PLA), poly(glycolic acid) (PGA), poly(lactide-co-gly-colide) (PLGA), and polyurethane, have been successfullyelectrospun for various tissue engineering applications suchas bone, cartilage, blood vessel, skin, and nerve.11–14,16

Several studies have shown that fiber structure and fiberdiameter of the electrospun substrates can influence cellbehavior and are important factors in the development ofmany types of regenerated tissues. Neuronal stem cellselongate more on PLA nanofibers as compared to microfib-ers.18 A study using MC3T3-E1 osteoblast-like cells showsthat the cell density increases with increasing diameters ofelectrospun PLA substrates in the absence of the osteogenicfactors and fiber diameter affects cell spreading, prolifera-tion, and differentiation.19 Kwon et al. reported that the ad-hesion, spreading, and proliferation of human umbilical veinendothelial cells were better on the 0.3- and 1.2-lm diame-ter fibrous meshes than on the 7-lm diameter fibrous meshof PLA-CL 50/50.20

Only a few studies have been reported on the fabricationof the electrospun pHEMA fibers. Pradny et al. investigatedthe influence of the polymer molecular weight and solutionviscosity on electrospun pHEMA.21 A reactive electrospin-ning process was developed to synthesize the cross-linkedpHEMA nanofibers directly from monomer solutions, bythermal formation of a precursor solution to achieve neces-sary viscosity followed by the photopolymerization ofoligomers in situ during the electrospinning process.22 Theso-formed individual nanofibers were studied for their elas-tic properties. Magnetite nanoparticles were incorporatedinto the electrospun pHEMA nanofibers for potential appli-cations in drug delivery.23 Nevertheless, a systematicapproach to control the diameter and morphology of theelectrospun pHEMA fibers has not yet been reported. It isalso not clear whether the electrospun pHEMA fibersremain low fouling in terms of the resistance to proteinadsorption. The potential applications of the electrospunpHEMA fibers have barely been explored. As a dual func-tional and hydrophilic material, we expect that the electro-spun pHEMA fibrous membrane resists protein adsorptionmeanwhile the abundance of hydroxyl groups on pHEMAallows effective conjugation of protein molecules in a non-fouling background. Such materials, if conjugated with bio-adhesive proteins, will interact with cells exclusivelythrough specific interactions, and thus can be potentiallydeveloped into biocompatible tissue engineering scaffolds.The dual-functionality and high surface-area-to-volume ratioalso make electrospun pHEMA fibers ideal substrates as af-finity membranes for highly selective and efficient biomolec-ular separation, which utilizes specific interactions betweenone kind of solute molecule and a second molecule or func-tional group immobilized on the fibrous membranes andrequires high resistance to nonspecific adsorption.

In this work, pHEMA was first synthesized via free radi-cal polymerization and subsequently electrospun into fi-

brous scaffolds. Controllable fiber diameters were achievedby varying the polymer solution concentration and the elec-trospinning flow rate. The as-spun fibers were thermallypost-treated to improve the shape integrity and structuralstability of the fibers in aqueous environments. The waterabsorption and protein adsorption behavior of the electro-spun pHEMA fibrous membranes were then characterized.The protein binding capacity of the chemically activated fi-brous membranes was studied using bovine serum albumin(BSA) as a model protein. The electrospun pHEMA scaffoldswere finally functionalized with type I collagen for fibroblastadhesion and proliferation.

MATERIALS AND METHODS

Materials2-Hydroxyethyl methacrylate (HEMA, ophthalmic grade, pu-rity >99%) and tetraethylene glycol dimethacrylate(TEGDMA) were purchased from Polysciences (Warrington,PA). Sodium metabisulfite and ammonium persulfate wereobtained from Sigma–Aldrich. PCL (Mw ¼ 80, 000) wasobtained from Dow Chemical Company (Freeport, TX). Etha-nol (absolute 200 proof) was obtained from Pharmoco-AAPER. Hydrogen peroxide, o-phenylenediamine (OPD), andpotassium bromide (IR grade) were acquired from Sigma–Aldrich. Sulfuric acid was purchased from EMD Chemicals(Gibbstown, NJ). Cyanogen bromide (CNBr) was supplied byAlfa Aesar (Ward Hill, MA). All chemicals were used asreceived. Human plasma fibrinogen (Fg) and BSA were pur-chased from Sigma–Aldrich. Horseradish peroxidase (HRP)-conjugated polyclonal goat anti-human fibrinogen wasobtained from USBiological (Swampscott, MA). Human typeI collagen was purchased from Advanced Biomatrix (SanDiego, CA). Bicinchoninic acid (BCA) protein assay kit wasobtained from pierce (Rockford, IL). Phosphate-buffered sa-line (PBS, pH 7.4, 10 mM, 138 mM NaCl, 2.7 mM KCl) andphosphate-citrate buffer (pH 5.0) were purchased fromSigma–Aldrich. Water used in the experiments was purifiedto a minimum resistivity of 18.0 MX-cm by a Barnstead fil-ter system. The human skin fibroblast cell line BJ, estab-lished from foreskin, was purchased from ATCC (Manassas,VA). Fetal bovine serum, penicillin–streptomycin, trypsin/EDTA (0.05%/0.53 mM), and VybrantVR MTT cell prolifera-tion assay kit were obtained from Invitrogen (Carlsbad, CA).

Preparation of pHEMAPHEMA was prepared via free radical polymerization, usinga similar procedure reported before.21 Briefly, ammoniumpersulfate (5.8 mg) and sodium metabisulfite (5.8 mg) wereadded into 9 mL of 66.3% (v/v) aqueous ethanol and mixedhomogeneously. The monomer HEMA (1.2 g) was thenadded. The polymerization was carried out at room temper-ature for 24 h.

Dynamic viscosityAfter the polymerization, the pHEMA solution prepared asdescribed above was diluted to different concentrations(Table I). The dynamic viscosity of these solutions was thenmeasured with a Haake Rotovisco series 1 rotational

456 ZHANG ET AL. ELECTROSPUN PHEMA

viscometer (Thermo Electron Corporation, Germany) equippedwith a cone-plate sensor system consisting of a rotatingC35/1� titanium cone and a stationary stainless steel PP35plate. With the fluid placed between the cone and plate, thecone was rotated for 60 s at a constant shear rate of 100 s�1.The steady-state viscosity of the pHEMA solutions wasdetermined.

ElectrospinningAn electrospinning process was used to generate pHEMAfibers. The experimental apparatus consists of a 5-mL sy-ringe with a blunt stainless steel 21G needle (BD Bioscien-ces, San Jose, CA), a syringe pump (SP101i, World PrecisionInstruments, Sarasota, FL) for controlling the feeding rates,a grounded rectangular aluminum foil covered collectorplate, and a high-voltage power supply (ES30P-5W, GAMMAHigh Voltage Research, Ormond Beach, FL). A 25 kV high DCvoltage was applied between the needle and the groundedcollector to initiate the jet. The distance between the needletip and the collector was set to 15 cm. The pHEMA solu-tions at different dilutions (Table I) were electrospun tostudy the effect of the polymer concentration. To investigatethe effect of the flow rate, the pHEMA stock solution P1was delivered with controlled flow rates ranging from 3.8 to80 lL/min, using the syringe pump. For the PCL electro-spun samples, a 10 wt % PCL solution was prepared inchloroform, stirred overnight, and electrospun at an injec-tion rate of 10 lL/min.

Scanning electron microscopy (SEM)The morphology of the electrospun samples was character-ized using an FEI Quanta 200 scanning electron microscope.Dried specimens were mounted onto aluminum stubs andcoated with a very thin layer of silver, using a K575X TurboSputter Coater (Emitech, United Kingdom) at 30 mA for 45s. The morphology was then characterized under high vac-uum conditions at an accelerating voltage of 20–25 kV. Theaverage diameter of the fibers for each sample was deter-mined from the SEM images using the ImageJ software. Atleast 10 fibers for each image and three images for eachsample were used for the analysis.

Thermal treatmentTo enhance the shape and structural integrity of the electro-spun pHEMA fibrous membranes, post-electrospinning heattreatment was carried out at 90�C for different lengths oftime. The percentage of mass loss for samples with or with-

out thermal treatment was measured to characterize theirstability in the aqueous environment. The pHEMA fibers,electrospun from P1 solution at a flow rate of 10 lL/minfor 4 h, were heated for 24, 48, or 72 h. The native or heat-treated membranes were punched into 15-mm diameterdisks and weighed. Samples were then transferred to 24-well plates with 0.5 mL water added in each well. After 1,2, 4, 8, 16 h and 3, 5, or 7 d, samples were withdrawn fromwater, dried, and weighed again. The percentage of the totalmass loss for each sample was then calculated as the ratioof the mass difference before and after water incubation tothe initial mass of the sample. SEM was used to analyze thesurface morphology of the untreated or heat-treated sam-ples after water incubation. Fourier transform infrared(FTIR) spectra of the native and heat-treated membraneswere obtained on a Digilab Excalibur FTS 3000 spectrome-ter (Digilab, Randolph, MA) in the wavenumber range of400–4000 cm�1 at a resolution of 4 cm�1. The electrospunmembrane (�1 mg) was ground with 100 mg desiccatedKBr and pressed into a pellet, of which absorbance FTIRspectrum was measured.

Water absorptionTo measure water absorption, the electrospun membraneswere immersed in water for 24 h. The hydrated samples weretaken out and weighed after removal of excess surface waterwith Kimwipe. The water absorption was calculated on thebasis of the weight difference of the membranes before and af-ter water uptake, using the following expression:

Water absorption ½%� ¼ Ws �Wd

Wd� 100%

where Ws is the weight of the membrane after uptake ofwater, and Wd is the initial weight of the dry membrane.

The water absorption behavior of pHEMA hydrogel wasalso measured for comparison. To prepare the pHEMAhydrogel, 1 g HEMA and 40 mg TEGDMA were dissolved ina solvent of distilled water and ethylene glycol (0.2 g/0.3 g),followed by the addition of 0.1 mL each of freshly preparedsolutions of 15% sodium metabisulfite and 40% ammoniumpersulfate. The mixture was poured between two clean glassplates separated by a 0.0381-cm thick Teflon spacer andpolymerized overnight. The polymer sheet was thenreleased from the glass plates and soaked in water with fre-quent changes to leach out unreacted monomers, initiatorsand oligomer residues.

TABLE I. Viscosity of pHEMA Solutions and Diameter of the Electrospun Fibers

Solution P1 P2 P3 P4

pHEMA stock solution (mL)a 4 2 1.33 166.3% aqueous ethanol (mL) 0 2 2.67 3Viscosity (Pa�s) 0.353 0.096 0.027 0.013Fiber diameter (nm)b 1025 6 384 396.8 6 44.7 269.7 6 26.4 N/A

aThe pHEMA stock solution was prepared by polymerizing 1.2 g HEMA, 5.8 mg ammonium persulfate, and 5.8 mg sodium metabisulfite in 9

mL 66.3% (v/v) aqueous ethanol for 24 h.bElectrospinning was carried out at a flow rate of 3.8 lL/min. N/A: not available.

ORIGINAL ARTICLE

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | 1 DEC 2011 VOL 99A, ISSUE 3 457

Enzyme-linked immunosorbent assay (ELISA)The adsorption of fibrinogen (Fg) onto the electrospunpHEMA membranes was measured by a direct ELISAaccording to the standard protocol as described below.24

The heat-treated electrospun membranes were first soakedin water for 24 h to leach out unreacted monomers, initia-tors, and oligomer residues, dried, and weighed. Sampledisks were then placed in individual wells of a 24-wellplate, washed with 500 lL of PBS, and incubated with 500lL of 1 mg/mL human Fg in PBS at 37�C for 90 min. Mem-branes were then rinsed with 500 lL PBS for five times andsoaked in 500 lL of 1 mg/mL BSA solution at 37�C for 90min to block the area unoccupied by Fg. After being rinsedwith PBS for five times, samples were incubated with 500lL of 5.5 lg/mL HRP-conjugated anti-Fg at 37�C for 90 min,followed by repeated PBS washing for five times. Mem-branes were subsequently transferred to new wells and 500lL of 0.1M phosphate-citrate buffer containing 1 mg/mLOPD and 0.03% hydrogen peroxide was added in each welland incubated for 20 min at 37�C to develop color. Thereaction was finally ceased by adding 500 lL of 1M sulfuricacid in each well and the absorbance of light intensity at490 nm was determined by a UV–visible spectrophotometer(UV-1601, SHIMADZU). PCL electrospun fiber was used asthe positive control in our experiments. PHEMA fibroussamples with no Fg or anti-Fg applied during the ELISA testwere used as the negative controls. The optical density at490 nm was normalized to the polymer mass. The mass-normalized absorbance from PCL fibers was set to 100%for calculating relative adsorption values of other samples.

Protein immobilizationTo study the protein binding capacity of the electrospunpHEMA, BSA, a model protein, was coupled to the CNBr-acti-vated pHEMA membranes following a previously reportedprocedure.25 The CNBr solution was prepared in water at 50mg/mL and adjusted to pH 11.5 with NaOH. The pHEMAmembranes were then reacted with the CNBr solution undergentle agitation for 60 min and washed with pH 11.5 waterthree times. Then, 0.5 mL of 1 mg/mL BSA solution, preparedin 0.1M NaHCO3 buffer containing 0.05M NaCl, was added toeach membrane and incubated with gentle shaking for 3 h atroom temperature. The BSA binding capacity of the electro-spun pHEMA membranes was calculated as follows:

q ¼ ðCi � CtÞV=m

where q is the BSA binding capacity (mg/g membrane), V isvolume of the BSA solution, Ci and Ct are the BSA concentra-tions in the initial and final solutions, and m is the drymass of the membrane before the CNBr activation. The BSAconcentration was determined by BCA method using aBCATM protein assay kit.

Cell cultureFibroblasts were maintained in continuous growth on tissueculture polystyrene flasks in ATCC-formulated Eagle’s mini-mum essential medium, supplemented with 10% fetal bo-

vine serum and 2% penicillin–streptomycin solution, at37�C in a humidified atmosphere containing 5% CO2. Topassage cells, cells were removed from the flask surfaces bywashing with 10 mL of PBS followed by incubation in 2 mLof trypsin/EDTA for detachment. After cells had detached,cells were resuspended in supplemented medium andreplated onto tissue culture polystyrene flasks. Cells werepassaged once a week.

The electrospun pHEMA fibrous membranes were firstchemically functionalized with human type I collagen tofacilitate cell adhesion. The 1-cm diameter membranes wereactivated with CNBr as described above and incubated withcollagen I overnight at 4�C. The collagen I solution was pre-pared in the NaHCO3/NaCl coupling buffer at 1 mg/mL andadjusted to pH 9.5 with NaOH.26 The pHEMA hydrogel diskswere also functionalized as control for comparison of cellbehavior. All collagen-modified pHEMA samples were thentransferred to a sterile 24-well culture plate, washed withsterile NaHCO3/NaCl coupling buffer three times, andblocked with 1 mg/mL sterile BSA for 30 min. Confluentfibroblasts were trypsinized, centrifuged, and diluted in sup-plemented medium at a final concentration of 100,000cells/mL. After the BSA blocking solution was removed andsamples were washed with PBS, 1 mL cell suspension wasadded to each well. The cells were then incubated with thesamples for 1 or 3 days at 37�C. Quantities of the adheredcells were estimated by measuring the conversion of water-soluble MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide] to insoluble formazan via a VybrantVR MTTcell proliferation assay kit following a standard protocol. Toimage cells on the electrospun membranes, samples werewashed with PBS, fixed in 2.5% glutaraldehyde overnight,washed with water, and dehydrated sequentially in 30, 50,70, 80, 90, 95, and 100% ethanol for 30 min each. SEMimages were then taken using an FEI Quanta 200 scanningelectron microscope.

RESULTS AND DISCUSSION

Polymer synthesis and viscosity measurementTo electrospin pHEMA, the monomer HEMA was first poly-merized into pHEMA by free radical polymerization accordingto Scheme 1 using redox initiators, ammonium persulfate andsodium metabisulfite, following a similar procedure reported

SCHEME 1. Preparation of poly (2-hydroxyethyl methacrylate) via free

radical polymerization.


before.21 The polymer was diluted in 66.3% ethanol to gener-ate solutions with different concentrations, denoted as P1–P4(Table I). Dynamic viscosity of these pHEMA solutions wasmeasured at a constant shear rate of 100 s�1. The stock solu-tion P1 has a viscosity of 0.353 Pa�s (Table I). When the stocksolution was diluted with solvent to half, one-third, orone-fourth of its original concentration, the solution viscositywas decreased to 0.096, 0.027, and 0.013 Pa�s, respectively.Changing the polymer concentration can vary the solutionviscosity.

Electrospinning of pHEMAPHEMA solutions with varied concentrations were electro-spun to study the role of polymer concentration on the gener-ated membranes. All electrospun membranes appear white.SEM images show that electrospun scaffolds at all concentra-tions possess a porous structure with randomly oriented non-woven fibers (Fig. 1). The stock pHEMA solution P1 leads tothe formation of smooth microfibers and nanofibers with an

FIGURE 1. SEM images of the pHEMA fibers electrospun from solutions of (a) P1, (b) P2, (c) P3, and (d) P4, with various polymer concentrations

at a feeding rate of 3.8 lL/min.

FIGURE 2. Linear relationship of pHEMA fiber diameters with viscos-

ity of the polymer solutions. The trendline shown has an R2 ¼ 0.998.

ORIGINAL ARTICLE


average diameter of 1025 6 384 nm (Figs. 1(a) and 2). Whenthe concentration decreases to half or one third, fibers appearthinner and the fiber diameter is more uniformly distributed,as compared to the scaffolds from P1, as shown in Figure 1.The diameter of nanofibers from P2 and P3 is 396.8 6 44.7nm and 269.7 6 26.4 nm, respectively (Fig. 2). In the case ofthe P4 solution with a lowest polymer concentration, thinnanofibers with beads-on-a-string structure were formed[Fig. 1(d)]. Fibers appear not uniform with rough surfaces.Direct dropping of polymer solution from the needle tip to thecollector was also observed (data not shown). Results showthat the concentration/viscosity of the polymer solutionsignificantly affects fiber diameter and morphology. Higherconcentrations lead to larger diameter of the fibers, while lowconcentration favors formation of fibers with beads. Figure 2indicates a linear dependence of fiber diameter on thesolution viscosity. Previous studies reported that diameter ofelectrospun fibers increased with solution concentrations ofpolymers for a variety of synthetic or natural materials suchas PGA,27 polydioxanone,28 elastin,12 fibrinogen,29 and colla-gen,12,30,31 which is consistent with our results. The beadformation when electrospinning the P4 solution is due to thelowest viscosity of the polymer solution. Formation of beadsand beaded fibers during electrospinning has been widelyobserved before and attributed to the capillary breakup of theelectrospinning jets by surface tension.14,32,33 One key factorinfluencing morphology of the electrospun end-product is thecompetition between the surface tension and viscoelasticforce of the polymer solution. Decreasing viscosity of the poly-mer solution favors the formation of beads while smoothfibers are more likely to be formed for the more viscous solu-tions. In the case of electrospinning the P4 solution, adecreased pHEMA concentration results in a low solution vis-cosity, thus the beaded fibers appears.

The effect of flow rate on electrospun fibers was alsoexplored. Flow rates ranging from 3.8 to 80 lL/min wereapplied to electrospin pHEMA stock solution P1. SEMimages show that smooth bead-free fibers were formed forall flow rates (Fig. 3). Variation in flow rate yielded signifi-cant changes in fiber size. Figure 4 shows that the averagediameter of fibers increases with the flow rate. It appearsthat the initial slope of the diameter/flow rate correlation atlow flow rates is greater than the slope at high flow rates.Actually, a power regression fits the data better with an R2

of 0.9833 compared with the linear regression with an R2 of0.9626. This might be due to the shear thinning behavior ofthe polymer, that is, reduction in viscosity at high rates ofdeformation. Polymer molecules are stretched out and dis-entangled at high rates of deformation, enabling them toslide past each other with more ease, hence, lowering thebulk viscosity of the solution. PHEMA fibers ranging from 1lm to 3.6 lm in diameter were produced. Besides intrinsicdifferences in fiber size, close examination of SEM imagesalso indicates difference in the morphology of electrospunpHEMA fibers prepared with various feed rates. Membraneselectrospun at a high rate of 80 lL/min exhibited fusion ofindividual fibers at the interconnected junctions, possiblyfacilitated by an incomplete removal of solvent by the time

the fiber jet reached the collection plate [Fig. 3(a)]. A flowrate higher than 80 lL/min led to direct dropping of poly-mer solution from the spinneret onto the collector.

Post-electrospinning thermal treatmentThe electrospun pHEMA membranes were heat-treated toenhance the structural integrity. Figure 5 shows the percent-age of mass loss of the untreated and heat-treated samples af-ter being soaked in water for different amounts of time. Forthe untreated membrane, the mass loss mainly occurred dur-ing the first 4 h with 16% of mass loss after 1-h incubationand 28.1% of loss after 4-h incubation [Fig 5(a)]. The samplemass remained unchanged afterwards. When the sampleswere thermally treated at 90�C for 24 h, the rate of mass lossin water was significantly reduced. Mass reduction mostlyhappened during the first 16 h. Approximately 12% of themass was lost after 7 days in water [Fig 5(b)]. The mass lossrate was further decreased if samples were heated for longer.Samples post-heated for 48 h and 72 h lost 8.3% and 7.3% ofthe total mass after 7-day incubation [Fig. 5(c) and (d)], whichmight result from the leaching of small amount of unreactedmonomers, initiators, and oligomers in the membranes.

Significant size reduction of the untreated sample diskswas observed after they were soaked in water. Figure 6(a)shows the surface morphology of an untreated sample after24 h incubation in water, demonstrating severely damaged fi-brous structure and reduced porosity, especially for thelayers closer to the membrane surface. The surface materialsof the membrane had easier access to bulk water thus maybe dissolved in water, causing surface fibers merging to-gether. To be further used in the biomedical applications suchas wound dressings, tissue scaffolds, and affinity membranes,the membranes need to maintain their shape and structuralintegrity, at least for a certain time period. Therefore, it isnecessary to improve the membrane integrity. For samplesheat-treated for 12 h, fiber morphology was mostly preservedafter water incubation [Fig. 6(b)]. Fibers appeared noticeablyswollen, even with a 72-h drying process after water incuba-tion, as compared with the as-spun fibers [Fig. 3(e)], whichmight be attributed to the strong association of hydroxylgroups of pHEMA with water. Results from mass loss experi-ments and SEM images both suggest that thermal treatmentfor 48 h or longer are sufficient to keep the integrity and fiberstructure of the electrospun pHEMA membranes.

To determine possible structural change of electrospunfibers caused by heating, FTIR spectra of the electrospunpHEMA fibers before and after thermal treatment weremeasured (Fig. 7). The absorbance ratio of C¼¼C (1636cm�1) to C¼¼O (1723 cm�1) decreased by 87% from 0.139for the prior-heat sample to 0.018 for the post-heat sample,indicating that the free radical polymerization was incom-plete in as-spun fibers and progressed further during theheating process. Weaver et al. reported that the degree ofpolymerization (DP) of pHEMA significantly affected itswater solubility.34 The HEMA oligomers with small DP werewater-soluble while pHEMA with DP greater than 50 wasinsoluble in water at room temperature. It is possible thatby thermal treatment the unreacted monomer and low MW


oligomer in the electrospun membranes were further poly-merized to form high MW pHEMA, which was not soluble inwater, leading to increased membrane integrity. Anotherpossible reason for the increased integrity is that when heatwas applied neighboring fibers may fuse together. Previousreports from Ramakrishna et al. showed increased struc-tural integrity of electrospun membranes by post-heat treat-ment, which was attributed to fusion of overlapping fibersat elevated temperatures or increased crystallinity afterannealing.35–37 Our differential scanning calorimeter results(data not shown) did not show obvious improvement ofcrystallinity for heat-treated fibers.

Water absorptionThe pHEMA fiber is a very hydrophilic material. Water dropcan be immediately sucked into the membrane once in con-tact with the surface. To characterize the hydration capacityof the membranes, the water absorption characteristics ofthe electrospun pHEMA membranes was determined basedon the weight difference of the membranes before and afterwater uptake relative to the dry mass of the membranes(Fig. 8). The fibrous membranes electrospun from thediluted P2 solution at a feeding rate of 3.8 lL/min (ES-3.8p2) had 96% water absorption. The membranes spunfrom P1 solution at 3.8 lL/min to 80 lL/min had water

FIGURE 3. SEM images of the pHEMA fibers electrospun with the flow rates of (a) 80 lL/min, (b) 60 lL/min, (c) 40 lL/min, (d) 20 lL/min, (e) 10

lL/min, and (f) 3.8 lL/min. The solution used for electrospinning is the pHEMA stock solution P1.

ORIGINAL ARTICLE


absorption of 200% or above. As a comparison, the pHEMAhydrogel only absorbed 70% water. It appears that the mem-branes with fiber diameter of 1 lm or above absorbed morewater than the nanofibrous scaffolds. The measured hydra-tion capacity may originate from the following three contri-butions: trapped water in the porous structure of the mem-branes, bound water molecules around the pHEMA fibers,and captured water molecules in the confined space betweenpHEMA chains. High surface-area-to-volume ratio of the elec-trospun fibers results in more bound water molecules on thefiber surface, whereas the porous structure of the scaffoldstraps water inside the pores, both of which contribute to theincreased water absorption of the fibrous scaffolds as com-pared with the pHEMA bulk hydrogel. With the increased

fiber diameter, there might be more water captured betweenthe neighboring pHEMA chains, further increasing the waterabsorption. Our pHEMA fibrous membranes exhibited waterabsorption up to 280%, whereas typical film wound dress-ings (e.g., BioclusiveVR ) only demonstrated water absorptionof 2.3%.16 Such fibrous membranes, if employed as wounddressings, will be able to absorb wound exudates muchmore efficiently than film dressings.

Protein resistance of electrospun pHEMAThe nonfouling property of the pHEMA electrospun fibrousscaffolds was assessed for their resistance to protein adsorp-tion by ELISA, using fibrinogen (Fg) as a model protein. Fg isa large protein (MW ¼ 340 kDa) commonly used to evaluatethe nonfouling characteristics of materials due to its abilityto easily adsorb to a wide range of materials and its roles inthe inflammatory response.38–41 Fg adsorption from bloodcan lead to platelet adsorption and blood clot formation

FIGURE 4. Correlation of pHEMA fiber diameters with flow rates of

electrospinning fitted to a power regression function. The trendline

shown has an R2 ¼ 0.9833.

FIGURE 5. Percentage of mass loss in water of electrospun pHEMA

fibers heat-treated at 90�C for (a) 0 h, (b) 24 h, (c) 48 h, and (d) 72 h.

[Color figure can be viewed in the online issue, which is available at

wileyonlinelibrary.com.]

FIGURE 6. SEM images of electrospun pHEMA fibers after 24 h of water incubation for (a) untreated fibers and (b) fibers heat-treated for 12 h.

All samples were dried for 72 h at 90�C before the SEM characterization.


when a material is exposed to blood.38,42 All pHEMA electro-spun samples for ELISA were thermally treated for 48 h toenhance structural integrity. PCL electrospun fiber, whichfacilitates protein adsorption and cell attachment and hasbeen widely investigated for tissue engineering applica-tions,11,43 was used as the positive control. The absorbancewas normalized to the sample mass to eliminate the influ-ence of mass difference among samples. The absolute absorb-ance for PCL electrospun membranes (�10 mg) at 490 nmwas 3.1 (data not shown), corresponding to a normalized ab-sorbance of 308.33 g�1 (Fig. 9). The mass-normalized ab-sorbance from PCL samples was set as 100% for calculatingthe relative adsorption. It can be seen from Figure 9 that pro-tein adsorption on electrospun pHEMA samples was signifi-cantly reduced as compared with that on PCL. The absoluteabsorbance for all pHEMA samples (5–20 mg) was <0.06.The mass-normalized absorbance of three pHEMA samples,electrospun from pHEMA solutions P1, P2, and P3, was 4.0g�1, 3.2 g�1, and 3.3 g�1, corresponding to the relative

adsorption amount of 1.3 6 0.2%, 1.0 6 0.1%, and 1.1 60.2%, respectively, using PCL as a reference. Relative adsorp-tion for two negative controls was 1.1 6 0.2% and 1.3 60.3%. Samples for negative control 1 and 2 are pHEMA1,with control 1 applying no Fg and control 2 skipping incuba-tion steps for both Fg and HRP-conjugated anti-Fg duringELISA. Results show that the amount of adsorbed protein onpHEMA samples was comparable with that on negative con-trols, indicating that the pHEMA electrospun fibrous mem-branes can highly resist nonspecific protein adsorption.

It is generally believed that water plays an importantrole in surface resistance to protein adsorption. For exam-ple, hydrophilic and neutral poly(ethylene glycol) forms ahydration layer via hydrogen bonds while zwitterions forma hydration layer via electrostatic interactions.44 PHEMApossesses an abundance of hydroxyl groups which can bindwater via hydrogen bonds. Furthermore, the intrinsic closepacking of pHEMA molecules in electrospun fibers leads to

FIGURE 7. FTIR spectra of electrospun pHEMA fibers before and after thermal treatment for 48 h at 90�C. The C¼¼C peaks (1636 cm�1) were

circled. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

FIGURE 8. Water absorption of pHEMA hydrogel and electrospun

pHEMA membranes with different fiber diameters. Numbers following

ES represent the flow rate of polymer solution during electrospinning.

The P1 pHEMA stock solution was used to generate all electrospun

membranes except ES-3.8p2 which utilized the P2 solution.

FIGURE 9. Human plasma fibrinogen (Fg) adsorption on different

pHEMA fibrous membranes measured from ELISA, with electrospun

PCL as a reference. Relative adsorption values (mean 6 standard devi-

ation %, n ¼ 4) are shown on the top of the columns. pHEMA1,

pHEMA2, and pHEMA3 denote fibers electrospun from pHEMA solu-

tions of P1, P2, and P3, respectively, at a flow rate of 20 lL/min. Ctrl1

is pHEMA1 with no exposure of Fg during ELISA. Ctrl2 is pHEMA1

with no exposure of both Fg and HRP-conjugated anti-Fg during

ELISA. All samples were heat-treated for 48 h before ELISA.

ORIGINAL ARTICLE


a high local concentration of hydroxyl groups which canbind water on the fiber surface and capture water betweenthe neighboring polymer chains. As expected, electrospunpHEMA fibrous membranes are capable of binding a signifi-cant amount of water molecules (Fig. 8) and therefore areexcellent nonfouling materials.

Protein conjugation on activated electrospun pHEMANot only the electrospun pHEMA fibers can be used as non-fouling materials to resist physical adsorption of proteins,but also they possess many hydroxyl functional groupswhich allow for covalent conjugation of protein molecules ina nonfouling background, thus considered as dual-functionalmaterials. BSA was used as a model protein in this work tostudy the protein bioconjugation onto the electrospunpHEMA membranes with different fiber diameters. As shownin Figure 10, content of the BSA immobilized onto the CNBr-activated pHEMA membranes was 75–120 mg BSA/g mem-brane, much higher than 5 mg/g membrane for the activatedpHEMA hydrogel control. The much higher protein bindingcapacities of the electrospun membranes are attributed tothe high surface-area-to-volume ratios and porous structureof the electrospun fibrous membranes. It is also found thatthe highest BSA binding capacity occurred at an intermedi-ate fiber diameter of 2.4 lm when the membrane was spunat a flow rate of 40 lL/min, which is different from our orig-inal expectation. As the fiber diameter increases, the totalsurface area of the fibrous membrane decreases, thus weexpected to see a decreasing trend of the protein bindingcapacity with the increasing fiber diameter before theexperiments. Decreased BSA binding capacity at the smallerfiber diameter end might be due to small pore sizes of themembranes (Figs. 1 and 3) which led to limited mass trans-port of the protein molecules into the membranes. The elec-trospun pHEMA membranes with high binding capacity ofprotein in a nonfouling background are expected to findpotential applications in affinity membranes for protein puri-fication which require high loading of ligands for specificinteractions and no nonspecific protein adsorption. Hydro-philic cellulose has been widely used in membrane separa-tion. Previous study of using electrospun cellulose nanofib-ers as affinity membrane took a two-step procedure toobtain hydrophilic regenerated cellulose nanofibers by firstelectrospinning nanofibers of hydrophobic cellulose acetatefollowed by treatment in alkaline solution to remove the ace-tyl groups, due to difficulties to find suitable solvents to dis-solve cellulose to prepare cellulose fibers directly by electro-spinning.45 Such prepared regenerated cellulose fibers,however, still had 18% of nonspecific BSA adsorption.45 Theelectrospun pHEMA fibers in our work are prepared with aone-step method, resist nonspecific protein adsorption, andhave high ligand loading, therefore highly suitable to be usedas affinity membranes for protein separation.

Fibroblast growth on collagen I-functionalized electro-spun pHEMA scaffoldsThe electrospun pHEMA scaffolds, if functionalized withcell-adhesive proteins, can also be used for tissue engineer-

ing applications. Figure 11(a,b) shows that fibroblastsadhered and spread on collagen I-modified pHEMA fibrousscaffolds. Cell filopodia appeared to attach and grow alongthe electrospun fibers. SEM images also show the existenceof some fine fibers with much smaller size compared withthe dimension of the electrospun fibers [Fig. 11(b)], mostlikely indicating the deposition of the native ECM by fibro-blasts. Compared with cell adhesion on the collagen I-modi-fied pHEMA hydrogel [Fig. 11(c)], there were much morecells adhered on the modified fibrous scaffolds, suggestingthat the fiber structure of the electrospun scaffolds stronglypromotes cell adhesion. MTT proliferation assay shows thatthe quantity of cells on collagen I-functionalized fibrousscaffolds after culturing for 3 days was much more thanthat after 1-day culture (Fig. 12), indicating that the func-tionalized electrospun fibrous scaffolds significantly pro-mote cell proliferation. It is also demonstrated in Figure 12that there were a lot more cells on the modified fibrousscaffolds than on the modified hydrogel. As compared withthe flat hydrogel, electrospun fibers mimic the fibrous andporous structures of the natural ECM, making them promis-ing candidates as tissue engineering scaffolds. Besides thescaffold morphology, high surface-area-to-volume ratio ofthe electrospun scaffolds, which increases the surface areaavailable for collagen I attachment, might also contribute tothe significantly enhanced cell attachment on the functional-ized electrospun scaffolds. As demonstrated in our work,the functionalized pHEMA electrospun fibrous scaffoldsgreatly support cell adhesion, spreading, and proliferation.

Further discussionThe high water absorption and strong resistance to nonspe-cific protein adsorption of the electrospun pHEMA fibrousmembranes may lead to their potential biomedical

FIGURE 10. BSA binding capacity of pHEMA hydrogel and electro-

spun pHEMA membranes with different fiber diameters. Numbers fol-

lowing ES represent the flow rate of polymer solution during

electrospinning. The P1 pHEMA stock solution was used to generate

all electrospun membranes except ES-3.8p2 which utilized the P2

solution.


applications as wound dressings, which can absorb exces-sive wound exudates, present a barrier to microbial inva-sion, and provide a semi-occlusive structure to alleviatepain. This work is also directly relevant to affinity mem-brane and tissue engineering applications. The abundant

hydroxyl groups of the electrospun pHEMA fibrous scaffoldsallow for effective ligand conjugation. Such functionalizedscaffolds can be used to purify protein or modulate cellbehavior while maintaining a clean scaffold backgroundwith eliminated nonspecific protein adsorption.

CONCLUSIONS

PHEMA is synthesized via free radical polymerization frommonomer and successfully processed into fiber forms byelectrospinning. Nanofibers and microfibers within therange of 270 nm to 3.6 lm in diameter are realized by con-trolling the polymer concentration and the electrospinningflow rate. Post-electrospinning thermal treatment signifi-cantly improves stability of the electrospun membranes inthe aqueous environment. The electrospun pHEMA fibrousscaffolds, capable of forming strong hydration layers sur-rounding fibers due to the abundance of hydroxyl groups inpHEMA and high local concentration of pHEMA in fibers, ex-hibit excellent resistance to nonspecific protein adsorption.Scaffolds with fiber diameters >1 lm absorb >200% waterrelative to the dry mass of the scaffolds. After CNBr activa-tion of the hydroxyl groups, dual-functional electrospunpHEMA scaffolds can be further functionalized with ligandsin a nonfouling background. A BSA binding capacity as highas 120 mg BSA/g membrane is realized at an intermediatefiber diameter. The pHEMA fibrous scaffolds functionalizedwith collagen I significantly promote fibroblast adhesion,spreading, and proliferation.

ACKNOWLEDGMENTS

The authors thank Prof. Jun Hu for his assistance with FTIRexperiments and Ms. Marcia Weidknecht for her assistancewith the viscosity measurement.

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ORIGINAL ARTICLE


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Dual-functional electrospun poly(2-hydroxyethyl methacrylate)

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