Surface-functionalized electrospun nanofibers for tissue engineering ...

1034 H.S. Yoo et al. / Advanced Drug Delivery Reviews 61 (2009) 1033–1042an electric field, the polymeric solution with a moderate viscosity valueforms the Taylor cone at the tip of an injecting device [1]. Whentheelectric voltage applied between the injector and the collector exceedsthe surface tension force of the Taylor cone, the charged jets areeventually sprinkled to the ground. During the travel of the fibrous jets,the solvent evaporates, generating a solidified nanofibrous structure onthe ground. These fibers stack to form a membranous mesh with a fiberdiameter of several hundred nanometers. Since fabricating a nanoscaledmatrix can be performed by a simple electrospinning method,electrospinning has been considered as a versatile method for preparingnanofibrous meshes for various biomedical applications. Bio-mimickingnanofibrous meshes are of great interest because of their structuralsimilarity to the extracellular matrix of biological systems such ascollagen fibers.In order to apply electrospun nanofibers in biomedical uses, theirsurfaces have been chemically and physically modified with bioactivemolecules and cell recognizable ligands after the electrospinningmethod; this subsequently provides bio-modulating or biomimeticmicroenvironments to contacting cells and tissues. A variety of functionalizationstrategies of electrospun nanofibers with bioactivemolecules including proteins, nucleic acids, and carbohydrates havebeen employed [2]. In this review, physical and chemical immobilizationmethods of bioactive molecules on the surface of variouspolymeric nanofibers are described, and their applications to drugdelivery and tissue engineering systems are introduced.2. Surface modification techniques of nanofiber meshVarious degradable and non-degradable synthetic nanofibers havebeen surface-modified with bioactive molecules for advanced biologicaland therapeutic applications. Synthetic polymers offer easierprocessability for electrospinning and more controllable nanofibrousmorphology than natural polymers. Water soluble natural polymersare often considered to be difficult to directly process into nanofibersdue to their unstable nature such as being vulnerable to processingconditions and having a weak mechanical property [3,4]. Due to theprocessing benefits of synthetic polymers, a wide variety of naturalpolymers having unique biological functions can be immobilized ontothe nanofibrous surface of synthetic polymers without compromisingbulk properties. Such biologically functionalized synthetic nanofiberscan direct enhanced cell specific phenotype and organization, becausetissue regeneration process is strongly involved with diverse biochemicalcues on the cell contacting surface. For sustained drug deliveryapplications, the electrospinning process enables a wide varietyof hydrophobic therapeutic agents to be directly incorporated withinthe bulk phase of nanoscale fibers for controlled release. For example, abiodegradable polymer solution containing hydrophobic anti-cancerdrugs such as paclitaxel was directly electrospun to produce drugreleasing nanofibrous mesh [5]. Alternatively, hydrophilic and chargedmacromolecular drugs such as proteins and nucleic acids were covalentlyand physically immobilized onto the modified surface ofnanofibrous mesh for modulating cellular functions. The electrospunnanofiber mesh possesses highly interconnected open nano-porousstructure with a high specific surface area, offering an ideal conditionfor sustained and local drug delivery [6]. Various surface modificationtechniques for applying synthetic polymer nanofibers to tissue engineeringand drug delivery are presented here (Fig. 1 and Table 1).2.1. Plasma treatmentPlasma treatment of polymer substrates has been commonlyemployed to tailor surface adhesion and wetting properties by changingthe surface chemical composition [7–10]. Appropriate selection of theplasma source enables the introduction of diverse functional groups onthe target surface to improve biocompatibility or to allow subsequentcovalent immobilization of various bioactive molecules. For example,typical plasma treatments with oxygen, ammonia, or air can generatecarboxyl groups or amine groups on the surface [11–14]. Avarietyofextracellular matrix protein components such as gelatin, collagen,laminin, and fibronectin could be immobilized onto the plasma treatedsurface to enhance cellular adhesion and proliferation [15–18]. Electrospunnanofibers composed of poly(glycolic acid) (PGA), poly(l-lacticacid) (PLLA), or poly(lactic-co-glycolic acid) (PLGA) were modified withcarboxylic acid groups through plasma glow discharge with oxygen andgas-phased acrylic acid [19]. Such hydrophilized nanofibers were shownto enhance fibroblast adhesion and proliferation without compromisingphysical and mechanical bulk properties. Air or argon plasma treatmenthas been widely used as a facile surface modification technique for manybiomaterials, since its surface hydrophilicity can be easily increased withconcomitant elimination of surface contaminants. For example, variouselectrospun nanofibers made of poly(ε-caprolactone) (PCL), PCL/hydroxyapatite, polystyrene, and silk fibroin were surface-modified byair or argon plasma, resulting in an improved cell adhesion and proliferation[11,20–22]. When PCL nanofibers were modified with argonplasma, enriched carboxylic acid groups could be produced on thesurface [23]. When the surface activated nanofibers were soaked in asimulated body fluid solution, the bone-like calcium phosphatemineralization occurred on the surface of nanofibers. This mineralizednanofibrous scaffold exhibited improved wettability with a biomimickingbone structure, indicative of potential application for bonegrafting.2.2. Wet chemical methodPartial surface hydrolysis of biodegradable aliphatic polyester filmsand scaffolds under acidic or basic condition has been widely used tomodify the surface wettability property or to create new functionalities[24–26]. This is based on the random chemical scission of esterlinkages on the polymer backbones located on the very surface,resulting in the surface generation of carboxylic and hydroxyl groupsfrom degraded, yet water insoluble polymer fragments. Since theplasma treatment for nanofibrous mesh cannot effectively modify thesurface of buried nanofibers deeply located in the mesh due to thelimited penetration depth of plasma in the nanopores, wet chemicaletching methods can offer the flexibility for surface modification ofthick nanofibrous meshes [27]. When biodegradable polymericnanofibrous meshes are surface-modified using the partial hydrolysismethod, a special care must be taken. The duration of the hydrolysisand the concentration of hydrolyzing agents are important to optimallyproduce surface functional groups only with minimallychanging the bulk property [27]. NaOH-treated PLLA nanofibrousmesh was also used for hydroxyapatite mineralization [30]. Sincecarboxylic acids can chelate calcium ions, surface-induced nucleationand growth of minerals were shown to be enhanced on the surfacemodifiedPLLA electrospun scaffold. PCL electrospun nanofibers werealso used to modify the surface of thin PCL membrane for generatingnano-topographical surface [29]. When the modified membrane wastreated with 5 M NaOH, wettability was dramatically enhanced,showing almost zero water contact angle due to the capillary action onthe highly rough surface. When NIH 3T3 cells were cultured on thesurface of the modified nano-topographical membrane, favorable cellmorphology and adhesion was observed on the modified surface,possibly due to the unique hydrophilic surface topography. A surfaceaminolysis method using diamine species as an alternative hydrolyzingagent for polyester nanofibers was also employed to ensurepositive surface charge as well as to create amine functionalizedsurface [27,30]. Electrospun poly(L-lactide-co-caprolactone) copolymer(PLLC) nanofibrous scaffold was surface-modified via aminolysis,followed by immobilization of a cell adhesive protein, human plasmafibronectin [31].When the aminolysis reaction was performed extensively for electrospunPLLA nanofibrous mesh, periodic degradation and fragmentation in

H.S. Yoo et al. / Advanced Drug Delivery Reviews 61 (2009) 1033–10421035Fig. 1. Surface modification techniques of electrospun nanofibers. (A) Plasma treatment or wet chemical method. (B) Surface graft polymerization. (C) Co-electrospinning.Table 1Surface modification techniques of electrospun polymers for biomedical applications.Surface modification method Electrospun polymer Surface immobilized agent Potential application ReferencePlasma treatment PCL Gelatin Blood vessel TE [14]PCL Soluble eggshell membrane protein TE and wound dressing [21]PCL Calcium phosphate Surface mineralization/Bone TE [23]PCL – Nerve TE [22]P(LLA-CL) Collagen Blood vessel TE [15,16]Polystyrene – 3D cell culture system [12]PLLA Laminin Neural TE [18]Silk fibroin – Cartilage TE [17]Wet chemical method NaOH hydrolysis PLLA HAp Surface mineralization/bone TE [28]PCL – TE [29]Aminolysis PLLC Fibronectin Esophagus TE [31]PLLA – TE [32]Surface graft polymerization Initiation Graft copolymer –Ce (IV) PET PMAA Gelatin Blood vessel TE [37]Plasma PLLA, PGA, PLGA PAA – TE [19]UV PU PVP – Antibacterial membrane [38]Co-electrospinningBlending agentPLLA HAp – Bone TE [42]PEO (SEE) 3 -PEO – Antibacterial membrane [43]PLGA PLGA-PEG-NH 2 RGD TE [82]Abbreviations: PCL, poly(ε-caprolactone); P(LLA-CL), poly(l-lactic acid)-co-poly(ε-caprolactone); PLLA, poly(L-lactic acid); PLLC, poly(L-lactide-co-caprolactone) copolymer;PET, polyethylene terephthalate; PGA, poly(glycolic acid); PLGA, poly(lactic-co-glycolic acid); PU, polyurethane; PMAA, poly(methacrylic acid); PAA, poly(acrylic acid); PVP, poly(4-vinylpyridine); PEO, polyethylene oxide; HAp, hydroxyapatite; SEE, Ser-Glu-Glu; PEG, poly(ethylene glycol); RGD, Arg-Gly-Asp; TE, tissue engineering.

1036 H.S. Yoo et al. / Advanced Drug Delivery Reviews 61 (2009) 1033–1042the individual nanofibers occurred to produce water-dispersible micronlengthnano-cylinders [32]. During the bulk aminolysis, transverselyfragmented cylindrical PLLA nano-materials were produced presumablyby developing degradation-induced crystal structures such as stackedlamellae. In addition, these nano-cylinders had substantially high amountof amine functional groups on their surface, enabling further surfacefunctionalization with bioactive molecules. Generally, biodegradablenanofibers, mostly characterized by extremely long, entangled, anddensely packed 2D structure, have limitation in tailoring a desiredmacroscopically porous 3D structure for tissue engineering applications.The relatively short, water-dispersible, and biodegradable nano-cylinderswith enriched surface-amine groups are expected to serve a 3Dnanofiber self-assembly scaffold for cell delivery.2.3. Surface graft polymerizationVirtually all types of synthetic biodegradable polymers retain theirhydrophobic surface nature, often requiring hydrophilic surfacemodification for desired cellular responses. Surface graft polymerizationhas been introduced not only to confer surface hydrophilicity, but also tointroduce multi-functional groups on the surface for covalent immobilizationof bioactive molecules for the purpose of enhanced celladhesion, proliferation, and differentiation [33–36]. The surface graftpolymerization is often initiated with plasma and UV radiationtreatment to generate free radicals for the polymerization. Electrospunpolyethylene terephthalate (PET) nanofibers were modified with poly(methacrylic acid) by graft polymerization in a mild condition withoutany structural damage in the bulk phase [37]. The PET nanofibers werepre-treated with formaldehyde to generate hydroxyls groups on thesurface. The subsequent oxidization of surface hydroxyl groups byCe (IV) produced free radicals, thereby initiating the polymerization ofmethacrylic acid monomers from the surface. The carboxylic acidmoieties on the surface were then conjugated to immobilize gelatin onthe nanofibers. The surface density of carboxylic acid was shown toincrease with reaction time and the monomer concentration. Forantibacterial applications, electrospun polyurethane (PU) nanofiberswere modified with poly(4-vinyl-N-hexyl pyridinium bromide) on thesurface [38]. In this study, the PU fibers were first treated with argonplasma, which produced surface oxide and peroxide groups. When theplasma treated PU fibers were immersed in a 4-vinylpyridine monomersolution with exposure of UV irradiation, poly(4-vinylpyridine) graftedPU fibers were successfully produced. Through quaternization of thegrafted pyridine groups with hexylbromide, the surface-modified PUfibers were endowed with antibacterial activities. The viability of GrampositiveStaphylococcus aureus (S. aureus) and Gram-negative Escherichiacoli (E. coli) after contact with the PU fibers was measured. Theantibacterial efficacy of the modified PU fibers for S. aureus and E. coliwere 99.999% and 99.9%, respectively after 4 h contact, indicating highlyeffective antibacterial activities.2.4. Co-electrospinning of surface active agents and polymersWhile the aforementioned surface modification methods are intendedto be used for prefabricated electrospun nanofibers, nanoparticlesand functional polymer segments can be directly exposed on thesurface of nanofibers by co-electrospinning with bulk polymers [39–41].For example, when PLLA solution blended with hydroxyapatite (HAp)nanocrystals was co-electrospun, HAp was exposed on the surface ofthe resultant fibers, giving rise to high surface free energy and lowwater contact angle [42]. These composite fibers exhibited a retardeddegradation rate as compared to pure PLLA fibers due to the internalionic bonding between ester groups in PLLA and calcium ions in HAp. Inaddition, a novel in-situ peptide bio-functionalization method driven byan electric field was developed [43]. Firstly, an antimicrobial peptide,with three repeating units of three anionic amino acids, serine, glutamicacid, and another glutamic acid (SEE) 3 , was terminally conjugated topolyethylene oxide (PEO). The addition of (SEE) 3 -PEO conjugate to PEOsolution decreased viscosity, but increased solution conductivity. Duringco-electrospinning of the PEO/(SEE) 3 -PEO blend solution, electricallypolarizable SEE segment had significant influence on fiber morphology.When the collector was connected as an anode, thick and inter-weldedfiber morphology could be observed due to the high flow rate of theblend solution under an electric field. Electric field driven surfaceorientation of the SEE segment was also confirmed. It was expectedthat other combinations of electrospinnable polymer and polarizablepolymer conjugate could be possible for in-situ fabrication of surfacebio-functionalized nanofibers.3. Target molecules loading on the surface of nanofibers3.1. Physical adsorption for drug deliveryDrug-friendly physical immobilization on the surface can beachieved by using surface-modified prefabricated nanofibrous meshesthat have an extremely high surface area to volume ratio, resulting inhigher drug loading amount per unit mass than any other devices. Theimmediate release of drugs from the nanofiber surface can enable faciledosage control of some therapeutic agents, suitable for some specificapplications such as prevention of bacterial infection occurring withinfew hours after surgery [44]. In addition, hierarchically organizedstructure such as drug-loaded nanoparticles adsorbed on the nanofiberscan allow unique drug releasing profiles, which nanofiber itself cannotachieve, in addition to the high drug loading capacity [45,46]. Fig. 2describes three different modes of physical drug loading method onthe surface of electrospun nanofibers for drug delivery application.Particularly, therapeutic proteins and nucleic acids were physicallyimmobilized onto the surface for controlled delivery. If they wereencapsulated within the bulk phase of nanofibers by co-electrospinningwith polymers for sustained release, their activities after the releasediminish because of harsh electrospinning processing conditions.Besides, it is extremely difficult to homogeneously dissolve suchcharged molecules with high molecular weight in an organic phase forelectrospinning process. Thus, bioactive charged macromolecules werereadily adsorbed on the surface-modified prefabricated nanofibrousmesh via specific and nonspecific interactions for local and sustaineddelivery. In this section, from the standpoint of innovative surfacedesign, we will focus the drug loading method on the nanofiber surfacefor biomedical applications.3.1.1. Simple physical adsorptionPhysical surface adsorption is the simplest approach for loading drugon the nanofibrous mesh. Generally, electrostatic interaction, hydrogenbonding, hydrophobic interaction, and van der Waals interaction canbe used as a driving force for surface adsorption [47]. A typicalexample is the use of specific interaction between heparin and growthfactor [48–51]. Heparin, a highly sulfated glycosaminoglycan, has strongbind affinity with various growth factors such as fibroblast growth factor(FGF), vascular endothelial growth factor (VEGF), heparin-bindingepidermal growth factor (HBEGF), and transforming growth factor-β(TGF-β). This approach offers preservation of biological activity bypreventing early degradation of growth factors. Heparin immobilizationof biomaterial surface and subsequent attachment of growth factor canbe the most efficient way for local delivery of growth factors andconsequent mitogenic induction [52]. Heparin functionalized electrospunfibers subsequently loaded with bFGF were developed [53]. Lowmolecular weight heparin (LMWH) was successfully incorporated toeither PEO or PLGA electrospun fibers as free LMWH or LMWH-poly(ethylene oxide) (PEO-LMWH) conjugate form. PEO nanofibersincorporated with LMWH were first prepared by electrospinning anaqueous solution containing PEO and LMWH. When the PLGA/PEO-LMWH blend solution was electrospun to fibers, the surface heparincontent slightly increased, as compared to PLGA/LMWH fibers with

H.S. Yoo et al. / Advanced Drug Delivery Reviews 61 (2009) 1033–10421037Fig. 2. Three modes of physical drug loading on the surface of electrospun nanofibers.significant degree of bFGF surface immobilization. This was mainlyattributed to the slower dissolution of PEO-LMWH conjugates from theelectrospun fibers. These surface-functionalized electrospun matricescould be a promising growth factor delivery vehicle for inducing localangiogenesis. Anisotropically aligned electrospun nanofibrous scaffoldsloaded with growth factor were prepared [54]. Initially, heparin wascovalently immobilized onto the surface of poly(L-lactide) (PLLA) nanofibersthat were aligned either during electrospinning process orthrough mechanical stretch after electrospinning. Then, laminin (anextracellular matrix protein) and bioactive bFGF could be simultaneouslyadsorbed on the nanofibers by specific heparin-mediatedinteraction. It was shown that the neurite outgrowth and cell migrationcould be induced by the alignment of nanofibers and also furtherenhanced by immobilization of biochemical cues (laminin and bFGF).The results suggested that the combination of locally delivered bioactiveagents and nano-sized surface topography could synergistically promotethe therapeutic efficacy in repairing damaged or malfunctionedtissues.One of the most often described applications of electrospun fibrousmesh is postsurgical anti-adhesion barrier [55–57]. The adhesion betweeninternal tissues and/or organs can normally occur after all typesof surgical procedure, often abdominopelvic surgery, leading to severeclinical complications including small bowel obstruction, secondaryinfertility in women, chronic debilitating pain, and inadvertent enterotomyat reoperation [58]. The adhesion is the consequence of woundhealing process often associated with tissue inflammation. The electrospunnanofibers can meet the requirement of such anti-adhesion barrierapplication by physically separating the wound site from an adjacentorgan or tissue and concomitantly delivering local therapeutic agentssuch as antibiotics. In fact, the surface-modified nanofibrous mesheshave been proven to be an efficient barrier for preventing postsurgicaladhesion. As an adhesion barrier, a commercial antibiotic drug, Biteral®,was adsorbed to the electrospun PCL nonwoven sheet [44]. To load thedrug, the drug solution was simply dropped on the PCL nonwoven sheetand left to be completely adsorbed. Due to the unique morphologicalfeature of electrospun fibers, such drug loading can be a verystraightforward and effective method. In in vitro release data, nearly80% of the initial burst was observed within 3 h and the release endedafter almost 18 h. A rapid drug release profile is highly desirable forpreventing infections at an early stage. The abdominal adhesion studywas performed in rat models with defects created at two sites in theabdominal cavity. Macroscopical and histological observations demonstratedthat the electrospun nonwoven sheet coated with antibioticsconsiderably reduced abdominal adhesions, also resulting in improvedhealing. It was suggested that this type of adhesion barrier could bemuch more suitable for clinical applications when the degradation rateof the PCL electrospun sheet was tailored to the healing rate of theinjured site.3.1.2. Nanoparticles assembly on the surfaceThe assembly of nanoparticles on the surface of nanofibers has beenattempted for a variety of applications such as electronic, catalysis, andsensor devices [59–62]. The large interfacial area of the nanofibers canenable the fabrication of high-performance devices. Such hierarchicalnano-structure can also be constructed using therapeutically orbiologically functional nanoparticles such as silver or hydroxyapatitenanoparticles [63]. In addition, any combination of both drug encapsulatedpolymeric nanoparticles and nanofibers can be possible formulti-functional performance. Such functional nanoparticles werereadily embedded within or adsorbed onto nanofibrous mesh byelectrospinning technique [64,65], offering the chance for loadinghighly concentrated nanoparticles onto the surface. Recently, aninteresting assembly design using nanoparticles and nanofibers wasintroduced for drug delivery applications. The nanoparticles-onnanofibershierarchical structure was created using attractive forcebetween opposite charges generated by electrospinning and electrospray[46]. It was reported that poly(methyl methacrylate) (PMMA)solution was electrospun and polystyrene (PS) solution was simultaneouslyelectrosprayed from two separate countercharged nozzles in aside-by-side fashion. Two oppositely charged electrohydrodynamicjets were encountered with neutralization, resulting in a compositestructure consisting of electrospun PMMA nano- or microfibersuniformly combined with PS nano- or microparticles. The molecularweight and the composition of the polymer, along with processingconditions including voltage and apparatus configuration, were shownto affect the consequent composite structure. It was suggested that ifthe target molecules could endure the electrohydrodynamic stress andthe polymer used could be electrospinnable and electrosprayable, anycomposition of nanoparticles-on-nanofibers structure could beachieved. When lipoic acid (antioxidant drug) or gold nanoparticles

1038 H.S. Yoo et al. / Advanced Drug Delivery Reviews 61 (2009) 1033–1042suspension were electrosprayed, electrospun PMMA fibers weresuccessfully modified with the corresponding composition in an insitumanner. It was also demonstrated that the resultant compositestructure appeared to be relatively stable and this nanofabricationsystem could offer a one-step surface modification method creating ahierarchical nano-structure.3.1.3. Layer-by-layer multilayer assemblyA versatile surface modification method that allows surface coatingwith thickness from a few nano to several micrometers through precisecontrol has been realized by layer-by-layer (LbL) polyelectrolytemultilayer assembly [66–70]. The method comprises of alternativelayer-by-layer deposition of polyanions and polycations principallydriven by an electrostatic force on charged substrates, resulting in selfassembledmultilayer coating or free standing film. This technique hasattracted considerable attention due to the ease of its synthesis,universality for any complex structure of substrate, and the possibilityof using any composition for the coating layer. While charged therapeuticcomponent (e.g., heparin and DNA) can be easily incorporatedinto the multilayer assembly, the incorporation of insoluble anduncharged drugs appears to be difficult. Recent approaches such asencapsulation of drugs into charged particles and prodrug modificationhave overcome the limitation of current preparation methods [71,72].Inaddition, other intermolecular forces including hydrogen bonding,metal-ligand complexation, covalent bonding, hydrophobic interaction,and molecular recognition have been proven to be available formultilayer assembly as a driving force [73]. Many bioactive agents andchemical drugs have been assembled for topical drug delivery applicationsmainly on planar substrates such as silicon wafer, quartz slides,and metal oxide due to the ease of their synthesis and analysis. Thepolyelectrolytes multilayer was deposited on the surface of electrospunPS fibers [74]. PS fibers were initially prepared by electrospinningand then the fiber surface was endowed with negative charges bysulfonation of phenyl groups. LbL assembly with poly(allylaminehydrochloride) (PAH) and poly(styrene sulfonate) (PSS) was conductedon the surface of electrospun fibers, followed by the dissolution of PScore fibers, producing hollow polyelectrolytes complex tubes. Di-blockoligonucleotides (polyA15G15 and polyT15C15) multilayers were alsobuilt-up based on the DNA hybridization mechanism by the LbLtechnique. Upon build-up cycles, the DNA multilayer grew in a linearmanner, showing a smoother surface compared with the fiber surfacecreated by PAH/PSS multilayer. Moreover, gold nanoparticles wereshown to be homogenously and densely assembled into the PAH/PSSmultilayer. It was suggested that this facile surface modification ofelectrospun fibers with synthetic polymers (PAH and PSS) andbiopolymer (DNA) could provide the opportunity for creating a varietyof drug releasing surfaces for biomedical applications.In another work, polyelectrolyte fibers consisting of two weakpolyelectrolytes, poly(acrylic acid) (PAA) and poly(allylamine hydrochloride)(PAH) were fabricated by electrospinning [75].Firstly,inorderto avoid the formation and precipitation of insoluble polyelectrolytecomplexes, PAA and PAH were mixed at around pH 2.1, where PAAis fully protonated, resulting in a homogeneous PAA/PAH mixture.Through electrospinning process, the resultant PAA/PAH fibers exhibiteda typical cylindrical structure and was insoluble in water at higherpH due to ionic crosslinking of each polyelectrolyte. The fibers werefurther crosslinked thermally at 140 °C to prevent dissolution in highsalt conditions. Methylene blue (MB, cationic molecule), a model drug,was electrostatically adsorbed onto the polyelectrolyte fibers, whichwere pre-treated with 0.05 M NaOH in order to impart a negative chargeto the fiber surface. The release profile of MB could be modulated bycontrolling the pH of the incubating solution. In a higher pH solution, theMB exhibited slow or no release, whereas in a lower pH solution, therelease of MB was accelerated. However, in a phosphate buffered saline(PBS) solution, the MB release could not be sustained due to the chargeshielding and swelling of electrospun fibers. To achieve sustainedrelease profiles, hydrophobic perfluorosilane covered the surface ofPAH/PAA fibers pre-adsorbed with MB via chemical vapor deposition oftrichloro (1H, 1H, 2H, and 2H-perfulorooctyl) silane, followed by heattreatment creating a hydrophobic surface network. This hydrophobiccoating could efficiently obstruct rapid diffusion of MB and successfullyoffered a sustained release up to more than 20 h. It was also demonstratedthat temperature-controlled release could be achieved byLbL multilayer coating with PAA and poly(N-isopropylacrylamide)(PNIPAAM) on the polyelectrolyte fiber surface. It is well known thatPNIPAAM in aqueous solution show reversible sharp phase transition inresponse to the change of temperature. At temperatures below 37 °C,PNIPAAM is under well-hydrated extended coil form, and is soluble inaqueous solution, whereas at temperatures above 37 °C, PNIPAAM isdehydrated to become insoluble. On the basis of the thermo-sensitivenature of PNIPAAM as well as the diffusion barrier function of themultilayer, the PAA/PNIPAAM multilayer on the fibers inducedtemperature responsive release profile of MB in conjunction withsustained release. The thickness of multilayer was also shown to influencethe release profile in a manner related with the length of thediffusion path. In this study, the release control could be achieved by avariety of approaches including modulation of pH in solution, hydrophobicsurface coating as a diffusion barrier, polyelectrolyte multilayercoating, and temperature control. The electrospun polyelectrolyte fiberscombined with stimuli-responsive polymers could be a promisingdrug delivery platform for modulating drug release rates triggered byexternal stimuli.3.2. Chemical immobilizationIn order to immobilize bioactive molecules on the surface ofelectrospun nanofibers, chemical modification must be done toproduce reactive functional groups as shown in Fig. 3. Chemicalimmobilization of bioactive molecules onto the surface of electrospunnanofibers is favored over physical immobilization in tissue engineeringapplications. Because the immobilized molecules are covalentlyattached to the nanofibers, they are not easily leached out from thesurface-modified nanofibers when incubated over an extendedperiod. However, it should be noted that partial inactivation of theimmobilized molecules can occur upon the covalent modificationwhen active sites are chemically modified.Primary amine and carboxylate groups were most extensivelyemployed to immobilize bioactive molecules onto the surface ofnanofibers. Upon activation of the carboxylic acid groups by 1-ethyl-3-Fig. 3. Common surface functional groups for immobilization of bioactive molecules.

H.S. Yoo et al. / Advanced Drug Delivery Reviews 61 (2009) 1033–10421039(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccimide(NHS), nanofibers were subsequently conjugated to primaryamine groups of bioactive molecules [14,76]. Carboxylic groups on thesurface of polymeric nanofibers containing different amounts ofpolyacrylic acid were employed for conjugation with collagen [19].In other studies, acrylic acid-immobilized nanofibers were conjugatedto amine groups to prepare aminated nanofibers [77]. Those nanofiberswere further employed for in vitro cultivation of umbilical cordblood cells [77,78]. A mixture of poly(acrylonitrile-co-acrylic acid) andpoly(acrylonitrile-co-maleic acid) was electrospun and then lipasewas immobilized onto the surface [79,80]. Carboxylic acids fromacrylic acid and maleic acid monomer units were reacted with theamine groups of lipase, and the immobilized lipase showed a decreasein enzyme activities compared to the native one in both cases. Poly(vinylidene fluoride) (PVDF) nanofibers were prepared and coupledwith poly(methacrylic acid) on the surface by plasma-induced graftcopolymerization [81].Functional amine groups including primary and ε-amine groups arealso great candidates for covalent modification of electrospun nanofibersbecause of their high reactivity. However, direct conjugation of bioactivemolecules on the surface of nanofibers exhibited several limitations incell adhesion and proliferation: cells cannot easily recognize thesebiological ligands because the immobilized ligands are not fully exposedon the surface, but often sterically buried within the mesh structure.Thus, several hydrophilic linkers between bioactive molecules of interestand nanofibrous mesh were introduced to promote cellular recognition.For this purpose, a hydrophilic polymer spacer, poly(ethylene glycol)(PEG), was chemically conjugated to various hydrophobic polymericsegments for electrospinning [82,83]. A di-block copolymer of PLGA-PEG-NH 2 was synthesized and electrospun to expose the terminal aminegroup of PEG segment in the aqueous medium. Electrospun nanofibrousmeshes displayed primary amine groups on the surface and those aminegroups were subsequently employed to immobilize cell adhesivepeptides. A cell adhesive peptide, Gly-Arg-Gly-Asp-Tyr (GRGDY) peptidewas covalently conjugated to surface-amine groups of PLGA nanofibersfor enhancing proliferation of NIH3T3 cells [82]. The amount ofimmobilized peptide increased when the blend ratio of PLGA-b-PEG-NH 2 /PLGA was increased. The surface conjugation of the RGD peptidesignificantly enhanced cell attachment as well as proliferation. Enhancedcell attachment was also attributed to high wettability, positive surfacecharge, and topography of the modified nanofibers. A similar approachwas also employed to immobilize various growth factors for woundhealing, where rhEGF was immobilized to PCL nanofibers via a PEGlinker [83].Themodified nanofibers played a critical role in maintainingkeratinocytic differentiation, suggesting reduced scar formation afterwound recovery. Moreover, the immobilized rhEGF retained the activityof keratinocytic differentiation even in a harsh condition of enzymaticdegradation. This study showed that the immobilized rhEGF retained acomparable activity to that of the native rhEGF due to its resistance inenzymatic degradation at the wound site. Lysozyme was covalentlyattached to PLGA nanofibers via a PEG linker [84]. A mixture of PLGAand a di-block copolymer of PLGA-PEG-NH 2 was electrospun and theresultant nanofibers were post-modified with lysozyme. Lysozymeimmobilized onto the surface showed comparable enzyme activitiescompared to that of native lysozyme. This surface functionalizationapproach based on co-electrospinning of amphiphilic di-block copolymershas several advantages. The amount of enzyme to be immobilizedonto the nanofibers could be controlled by changing the blending ratio ofPLGA/PLGA-PEG-NH 2 in the electrospinning solution. Because a hydrophilicPEG segment with a terminal amine group in the block copolymerwould be preferably oriented on the surface, the degree of immobilizationwas proportional to the amount of the block polymer. Direct enzymeimmobilization on the nanofibers without using a PEG spacer could notretain these activities. For example, immobilized lipase on acrylic acidmodified electrospun nanofibers showed reduced enzyme activity to37.6% after immobilization [79]. In another case, immobilized chymotrypsinshowed decreased activity as low as 65% [85]. However, as shownin the conjugation of lysozyme and rhEGF to electrospun nanofibers, ahydrophilic linker played a significant role in maintaining the bioactivitiesof immobilized molecules. Chitosan was frequently electrospunfor chemical immobilization of bioactive molecules onto the surfacebecause of its functional amine groups. A copolymer of chitosan and poly(ethylene oxide) was electrospun to fibers and primary amine groups ofchitosan was crosslinked with glutaraldehyde [86]. The crosslinkednanofibrous mat showed an increase in the tensile strength, as comparedto that of unmodified nanofibers, and the degree of mechanical strengthwas proportional to the exposure time to glutaraldehyde gas.Hydroxyl groups were also employed for the chemical immobilizationof bioactive molecules. Modified polystyrene with a hydroxylcontaininginitiator was electrospun and alpha-chymotrypsin wascovalently attached on the surface [85]. Electrospun regeneratedcellulose nanofibers were functionalized with protein A/G for IgGpurification [87]. The nanofibrous membrane was treated with NaIO 4for activation of hydroxyl groups in the membrane. Then, protein A/Gwas added for covalent modification. No morphological change wasobserved after the modification.4. Applications4.1. Drug deliveryEmploying electrospun nanofibers as drug delivery vehicles has beenbased on their unique functionality and inherent nanoscale morphologicalcharacteristics [88].Inaddition,duetotheflexibility of its materialprocessing option, a variety of structural architectures containing drugmolecules could be fabricated from monolithic nanofibers to variousmultiple composition systems [89]. These important benefits allowfinely tuned drug eluting profiles that rely on controlling drug travelinglength or modulating the affinity between matrix materials and drugs.Drug release mechanism is associated with polymer degradation andcomplicated diffusion pathway along nano-void spaces within nanofibermesh. It has been shown that drug release profiles can be tailored byvarious formulation conditions such as polymer property, combinationof different polymers, surface coating, and the state of drug molecules ina solid phase [90–93].Hollownanofibrous tubes by coaxial electrospinningalso provided a promising structure for the encapsulation of targetdrug molecules [94]. This approach succeeded in achieving high drugloading and facilitation of the solubilization of some insoluble andintractable drugs. A rich variety of therapeutic agents such as antibiotics[55], anti-cancer drugs [5], polysaccharides [95], proteins[96], andgrowth factors [53] have been physically or chemically formulatedwithin the bulk phase of electrospun nanofibers or on their surface foraccomplishing controlled topical release within the defined period oftime. Such medicated nanofibers could be applied to various purposesincluding tissue engineering scaffolds, wound healing materials, andabdominal anti-adhesions after surgical procedure. While previousapproaches mainly focused on the encapsulation or embedment ofdrugs within the nanofiber bulk phase, recently introduced surfacemodifieddesigns for drug loading open up the new possibility ofconstructing more sophisticated drug delivery platforms. Electrospunnanofibers for drug and gene delivery application have been used fortissue engineering to improve therapeutic efficacy (Fig. 4). In addition,the fibrous surface structure shows strong adhesiveness to mucouslayers because their nano-porous structures instantly absorb moistureat mucous layers through nano-void volumes [97]. The superioradhesiveness toward biological surfaces allows nanofibers to be anideal candidate for topical drug delivery devices.4.2. Tissue engineeringVarious types of cells, including mesenchymal stem cells, embryonicstem cells, keratinocytes, and hepatocytes, cultivated on nanofibrous

1040 H.S. Yoo et al. / Advanced Drug Delivery Reviews 61 (2009) 1033–1042Fig. 4. Tissue engineering application is often combined with drug delivery strategy.meshes showed superior viability compared to other tissue engineeringmaterials probably because of the high surface/volume ratio ofnanofibers that mimic an extracellular matrix structure [98]. Galactosylatedmoieties were immobilized onto the surfaces of highly porousnanofibers for in vitro culture systems of rat hepatocytes [99]. Acrylicacid was photo-irradiated on polymeric nanofibrous meshes andcarboxylic acid groups were subsequently employed to conjugategalactopyranoside. This galactosylated scaffold composed of poly(εcaprolactone-co-ethylethylene phosphate) (PCLEEP) promoted cellularfunctions of rat hepatocytes when the hepatocytes were cultivated intospheroid-like aggregates. In another study, electrospun PCL nanofiberswere surface-modified with calcium phosphate [100], showing that thenanofibers strongly enhanced osteoblastic differentiation and proliferationfor a prolonged period of cell culture. The calcium phosphate layerson nanofibers showed similar characteristics of human bones, suggestingpotential application in bone tissue engineering. Hematopoieticstem cells were cultivated on the nanofibers surface-modified withhydroxyl, carboxyl, and amino groups [77]. It was found that aminatednanofibers most effectively promoted expansion of stem cells andprogenitor cells including CD34(+) and CD45(+). Endothelial cellswere cultivated on surface-modified poly(L-lactic acid)-co-poly(εcaprolactone)(P(LLA-CL)) nanofibrous meshes [16]. Electrospun polymericnanofibrous meshes were fabricated in two different ways;aligned and randomly distributed. These aligned and randomlydistributed nanofibers were subsequently modified with collagen onthe surface. Human coronary artery endothelial cells proliferated in thesame direction as that of the aligned surface-modified nanofibers. Theendothelial cells on the nanofibers showed high retention of theiroriginal phenotypes irrespective of the degree of alignment. In anotherstudy, human mesenchymal stem cells were cultivated on nanofibrousscaffolds for subcutaneous implantation of reconstructive surgery [101].In the study, stem cells were differentiated into chondrocytes on thenanofibrous mesh in bioreactors for efficient proliferation. It wasconfirmed that high expressions of collagen type 2 and aggrecan wereattained after 42 days of in vitro cultivation. Another good example ofapplication of surface-modified nanofibers to tissue engineering is theimmobilization of cell adhesive molecules including RGD peptides,fibronectin, and collagen. High surface areas of nanofibrous meshesenabled cell adhesive molecules to be immobilized onto the surface to agreater extent, when compared to conventional tissue engineeringscaffolds. This consequently increased cell adhesion on the surface andpromoted proliferation of the cultivated cells [82]. Cell adhesionproteins were also conjugated to electrospun nanofibers for cultivationof epithelial cells [31]. Another cell adhesive molecule, fibronectin, wasgrafted on the fibrous meshes composed of PLLC by glutaldehydecrosslinking. The morphology of the surface-modified nanofibers wassame compared to that of the unmodified ones. They also testedmechanical properties of the nanofibers, which showed a minordecrease in the strain strength after polyester aminolysis. Porcineesophageal epithelial cells cultivated on the nanofibers showedenhanced proliferation, which was confirmed by SEM, immunehistology,and protein analysis. Collagen was also employed forenhancing attachments and proliferation of stem cells [76]. Chemicallyimmobilized collagen on nanofibrous meshes strongly promoted cellgrowth rates as well as differentiation of neural stem cells in a dosedependentmanner. This was attributed to increase in the attachmentand preserved viability of cultivated stems cells on the collagennanofibrous meshes. Human mesenchymal stem cells were cultivatedin electrospun nanofibrous mesh composed of biodegradable polymer,PLGA beads and PLLA [102]. Histological analysis showed that thecultivated stem cells differentiated into osteocytes and chondrocytes.This study suggested that nanofibrous meshes with specific surfacemorphologies differentiated mesenchymal stem cells into specific typesof cells. Two layers of surface-modified nanofibrous meshes wereemployed for cultivation of hepatocytes [103]. The first fibrousmeshes were modified with galactose, while 3-methylcholanthrenewas loaded onto the second meshes for enhancing enzyme activity ofhepatocytes. The galactosylated layer increased attachments ofhepatocytes while the later layer played a role of a depot system ofthe bioactive molecule (3-methylcholanthrene). Currently, manyapproaches toward tissue engineering applications have beenlimited to in vitro studies, and only few studies were performed forin vivo application because cells could not be loaded within thenanofibrous meshes in a large quantity. This limitation can be overcomeby employing multi-layered nanofibrous scaffolds, where layers of cellsare proliferated in-between the layers of meshes. Thus, the cell layerscan proliferate and differentiate according to the microenvironment ofsurface-functionalized nanofibrous membranes. Various methods tofabricate 3D nanofibrous scaffolds have been attempted for in vivotissue engineering applications.5. ConclusionsElectrospinning of nanofibers is becoming a hot issue in biomaterials.In order to obtain high functionalities of electrospun nanofibers, thesurface of electrospun nanofibers was modified in various ways. Surfacesof nanofibrous meshes can be modified by plasma treatment, wetchemical method, surface graft polymerization, and co-electrospinning.For loading drugs on the surface for sustained release, drug encapsulatednanoparticles or another drug embedded layer can be assembled. Fortethering cell recognizable ligands on the surface, ligand molecules canbe chemically immobilized after functionalization. Surface engineerednanofibrous meshes are expected to have high potentials for drug andgene delivery and tissue engineering applications.

H.S. Yoo et al. / Advanced Drug Delivery Reviews 61 (2009) 1033–10421041AcknowledgementsThis study was supported by the grants from the National ResearchLaboratory Program of the Ministry of Education, Science and Technology,and the Korea Research Foundation (KRF-2006-005-J04602),and the Polymer Technology Institute, Sungkyunkwan University,Republic of Korea. The authors are grateful to Hayeun Ji for proofreadingthe manuscript.References[1] J. Doshi, D.H. Reneker, Electrospinning process and applications of electrospunfibers, J. Electrost. 35 (1995) 151–160.[2] Q.P. Pham, U. Sharma, A.G. Mikos, Electrospinning of polymeric nanofibers fortissue engineering applications: a review, Tissue Eng. 12 (2006) 1197–1211.[3] N. Bhattarai, Z.S. Li, D. Edmondson, M.Q. Zhang, Alginate-based nanofibrousscaffolds: structural, mechanical, and biological properties, Adv. Mater. 18 (2006)1463–1467.[4] T.G. Kim, H.J. Chung, T.G. 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