Arch. Pharm. Res. (2014) 37:69–78 DOI 10.1007/s12272-013-0284-2

REVIEW

Therapeutic applications of electrospun nanofibers for drug delivery systems Young Ju Son • Woo Jin Kim • Hyuk Sang Yoo

Received: 9 September 2013 / Accepted: 29 October 2013 / Published online: 15 November 2013 Ó The Pharmaceutical Society of Korea 2013

Abstract Electrospun nanofiber drug delivery systems have been studied using various techniques. Herein, we describe the fabrication of a drug-incorporating nanofiber. Drugs, such as proteins, peptide, antibodies, and small molecule drugs, can be loaded within or on the surface of nanofibers according to their properties. Hydrophobic drugs are directly dissolved with a polymer in an organic solvent before electrospinning. However, it is preferred to surface-immobilize bioactive molecules on nanofibers by physical absorption or chemical conjugation. Especially, chemically surface-immobilized proteins on a nanofiber mesh stimulate cell differentiation and proliferation. Using a dual electrospinning nozzle to create nanofiber sheet layers, which are stacked on top of one another, the initial burst release is reduced compared with solid nanofibers because of the layers. Furthermore, hybridization of electrospun nanofibers with nanoparticles, microspheres, and hydrogels is indirect drug loading method into the nanofibers. It is also possible to produce multi-drug delivery systems with timed programmed release. Keywords Nanofiber
Electrospinning
Drug delivery
Tissue engineering

Y. J. Son
H. S. Yoo Department of Biomedical Materials Engineering, Kangwon National University, Chuncheon 200-701, Republic of Korea W. J. Kim Department of Internal Medicine, School of Medicine, Kangwon National University, Chuncheon, Republic of Korea H. S. Yoo (&) Institute of Bioscience and Bioengineering, Kangwon National University, Chuncheon, Republic of Korea e-mail: [email protected]

Introduction Drug delivery systems using electrospun nanofibers have been developed in the last number of decades for application in many areas, such as tissue scaffolds, anti-bacterial sheets, and anti-cancer therapy (Lee et al. 2012; Tambralli et al. 2009; Wang et al. 2013). Although nanofibers alone have many advantages, such as high surface to volume ratio, porosity, and a structure that mimics the extracellular matrix (ECM) structure, the production of flexible sheets and loading of drugs has improved the function of nanofibers. Hydrophobic polymers such as poly(e-caploractone) (PCL) and poly(urethane) (PU) are easy to electrospin and exhibit high mechanical strength and elastic properties after electrospinning (Geun Hyung 2008; Lim et al. 2008). However, the hydrophobicity of electrospun nanofibers is not good for cell attachment and the nanofibers must be further modified to add hydrophilicity (Li et al. 2003). Nanofiber scaffolds have also been improved by loading proteins within/on the nanofiber mesh (Choi et al. 2008; Jia et al. 2013; Kim and Park 2006). Successful drug loading into the nanofiber for sustained release has been a key parameter for investigation. The high surface to volume ratio of the nanofiber is a unique structural feature; however, it causes an initial burst release of drugs (Verreck et al. 2003a; Kim et al. 2004; Yan et al. 2009). According to type of drug incorporated, such as small molecule drugs, proteins, and genes, the loading of the drug may require a different approach to obtain a successful release profile. Drugs can be simply mixed with the polymer solution, physically or chemically surfaceimmobilized, or indirectly loaded onto the nanofiber. The development of single nozzle to co-axial nozzle electrospinning could support the fabrication of a solid nanofiber that can control the initial burst release, overall release

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Fig. 1 Schematic diagram of simple drug loading method within the nanofiber or on the surface of the nanofiber and its release kinetics

kinetics, and multi-drug loading properties (Liao et al. 2006; Zhang et al. 2006). Furthermore, other types of biomedical devices, such as nanoparticles, microspheres, liposomes, and hydrogels can be hybridized to nanofibers to optimize the properties for specific applications (Kim and Yoo 2010; Ionescu et al. 2010; Mickova et al. 2012). This review focuses on various methods of drug loading and their advantages and limitations. We also summarize the different applications according to the methods of drug loading.

Simple drug loading In drug delivery systems, drugs can be loaded to the nanofiber by the processing of co-electrospinning or surface immobilized as shown in Fig. 1. Characteristics of drugs and the application of the device decide drug loading method. According to drug loading method within/on the nanofiber, it is expected different drug release process. Therefore, it is focused on summary two different drug incorporation skills, characteristic of drugs, and its application in this section.

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Co-electrospinning of drugs and polymer The simplest method of drug incorporation to nanofibers is to dissolve the drugs and polymer in the same solvent and coelectrospin. The incorporation of drugs within nanofibers exhibit high drug-loading efficiency in combination with self-assembled nanoparticles or microspheres that are welldispersed within the fibers and sheets (Verreck et al. 2003b). The first report describing drug delivery from an electrospun polymer was by Kenawy et al. (2002). Poly(ethylene-covinyl acetate) (PEVA) and poly(lactic acid) (PLA) were coelectrospun with tetracycline hydrochloride to fabricate a tetracycline-PEVA monolithic fiber for controlled release of tetracycline. The correct concentration of PEVA/PLA blending trapped tetracycline within and improved the initial burst release. However, over 50 % of drugs were released out within 5 days. Numerous drugs have been co-electrospun, including small molecule drugs, antibiotics, and proteins (Zeng et al. 2005a; Pattama et al. 2006; Kim et al. 2004). Drug solubility in the polymer solution is one of the main factors deciding the drug loading method (Zeng et al. 2005b). Hydrophobic drugs, such as doxorubicin and paclitaxel have electrospun well with organic solvents (Xu et al.

Electrospun nanofibers for drug delivery systems

2008, 2009) Hydrophilic drugs, such as peptides and proteins have been dissolved in aqueous phase with poly(vinyl alchol) (PVA) or PEO and uniformly electrospun (Gatti et al. 2013; Tang et al. 2012). However, it was proposed that the exposure of drugs to organic solvents and high voltage, can be harmful for drugs especially bioactive molecules (Yang et al. 2008). Furthermore, most simple physical mixing drug-loading methods exhibit short release profiles within several days (Meng et al. 2011; Luong-Van et al. 2006; Yu et al. 2013). This is because drug release in the initial stages is a result of simple diffusion, while in the later stages it is polymer degradation. In particular, drugs located around the nanofiber surface are released in the burst phase owing to the high surface-tovolume feature of the nanofibrous structure. Surface immobilization on the nanofiber Physical and chemical surface immobilization of drugs can avoid drug denaturation caused by high voltage or organic solvents, while taking advantage of the unique feature of the high surface to volume ratio of nanofibers. It is also possible to control the amount of drugs immobilized by controlling the by drug feeding ratio. During physical absorption, electrostatic interactions, hydrophobic interactions, hydrogen bonding, and van der Waals interactions are the main forces that retain the drugs on the nanofiber (Yoshida et al. 2006). Heparin, a negatively-charged polysaccharide, has a strong binding affinity for growth factors, such as basic fibroblast growth factor (bFGF), vascular endothelial growth factor (EGF), transforming growth factor-b (TGF-b), and heparin-binding epidermal growth factor (HBEGF) (Rajangam et al. 2006; Yayon et al. 1991; Lee et al. 2007; Sasisekharan et al. 1997). Therefore, heparin is useful for physically immobilizing growth factors on the surface of nanofiber meshes. PCL/ gelatin nanofibers were surface-immobilized with heparin bound platelet-derived growth factor-BB (PDGF-BB) (Lee et al. 2012). First, heparin was chemically immobilized to the surface-exposed amine of PCL/gelatin nanofiber, following which PDGF-BB was trapped by heparin on the nanofiber. As a result, the PDGF-BB release kinetics from heparin-immobilized nanofibers was dramatically reduced compared with the non-heparin-treated nanofibers, and was continuously released for 20 days. Thus, heparin conjugation controlled the release kinetics of PDGF-BB. Chemical modification of the nanofiber surface with amine, carboxyl, hydroxyl, or thiol groups, is another method for immobilizing drugs. Hydrophilic polymers, such as poly(ethyleneglycol) (PEG) and poly(ethyleneimine) (PEI) were co-electrospun with PCL and PU to maintain the mechanical strength of the mesh and provide the mesh with functional groups. Chemical conjugation methods are better

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for finely controlling the amount of incorporated drug on the nanofiber mesh than physically trapping the drug on the surface, and exhibits slow drug release kinetics with reduced initial burst release (Cho et al. 2010). Successful loading and release of a drug using functionalized hydroxyl polymer on nanofibers was introduced as a gene delivery system. Kim and Yoo (2010, 2013a, c) developed a gene-incorporated electrospun nanofiber mesh for wound healing. PCL-PEG nanofiber surface was modified with linear PEI to the end of PEG via a matrix metalloproteinase (MMP)-cleavable peptide, which was cleaved by MMPs in wound site. Plasmid DNA or small interfering RNA (siRNA) chemically conjugated to positively-charged amine PEI on the surface of the nanofiber according to the N/P ratio exhibited a successful release profile and gene transfection in the wound site. Surface-immobilized molecules do not always need to be released from the matrix. The ECM mimicking structure of nanofibers, whether random or aligned, have been studied for the purpose of stem cell differentiation into osteoblasts and neurons, even without any induction reagents (Ghoroghi et al. 2013; Schofer et al. 2009; Fang et al. 2010; Zhang et al. 2012a). The addition of bioactive molecules immobilized on the scaffold could enhance these effects (Pritchard et al. 2010; Jeong et al. 2010). Proteins, peptides, antibodies, and carbohydrates have been fixed on scaffold via chemical conjugation to surface exposed functional groups, such as carboxylate, thiol, and primary amine groups (Chan et al. 2009; Chua et al. 2005; Kim and Park 2006; Tigli et al. 2010). Cho et al. surface-immobilized nerve growth factor (NGF) on aligned nanofibers for the neuronal differentiation of mesenchymal stem cells (MSCs). Chemically-immobilized NGF was not released from the mesh over 7 days, and MSC differentiation into neuronal cells was effective owing to the synergic effect of the aligned nanofibrous structure and immobilized NGF on the nanofibers. Sheath nanofiber The limitation of drug release kinetic of the nanofiber with simple drug loading method is possible to be improved by introducing sheath layer on the solid fiber or mesh as shown in Fig. 2. Co-axial electrospinning technique has been developed for core/sheath nanofiber with drug within core of the nanofiber. Also, multi-layer stacked nanofiber was developed with subsequent electrospinning of two different solutions. Co-axial nanofiber Electrospinning can be modified for loading multiple drugs into a device and individually control the release kinetics of each drug. A co-axial needle is a horizontal arrangement

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Fig. 2 Schematic diagram of drug loaded nanofiber with sheath layer and its drug release kinetics

of outer and inner needles that separate two different solutions. It is easy to electrospin two immiscible polymer solutions containing drugs in the core and sheath because the two components are simultaneously electrospun from separate capillaries (Wang et al. 2012; Yu et al. 2011). Most of the core/sheath nanofibers used in drug delivery systems are loaded with proteins in core by co-electrospinning with the inner solution (Yu et al. 2013; Kim et al. 2008b; Song et al. 2013). In comparison to co-electrospinning of a drug with a single nozzle, the drug-loading efficiency is still high and the initial burst release property is decreased using sheaths of nanofibers. Hydrophobic shell materials, such as slow-degrading PCL, act as a barrier to simple diffusion of a core-loaded drug (Song et al. 2013). Drug release occurs after swelling or dissolving of the core polymer resulting in the formation of pores in the shell after dissolution of hydrophilic portion in core (Liao et al. 2006). Multi-drug delivery systems are also possible using co-axial electrospinning. Various drugs can be loaded by the co-axial system using two drug-loading methods, coelectrospinning by simple mixing and chemical surface modification, as explained in the previous section. Therefore, using two different methods the scaffold exhibits two independent drug release kinetics. In our previous study, we developed two different growth factor-loaded co-axial nanofibers (Choi and Yoo 2010b). The inner component

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was fabricated by electrospinning in the aqueous phase with bFGF to prevent denaturation of the protein in the organic solvent of the outer solution. After electrospinning, EGF was chemically conjugated to surface exposed amines on the mesh. Drug release kinetics of the core/shell nanofiber exhibit reduced initial burst release of core-loaded drug by prevention of the initial diffusion of the drug by the shell layer, which was maximized up to 90 % in 7 days. Furthermore, shell immobilized protein was rarely released. Therefore, released bFGF and surface-immobilized EGF stimulated enhanced cell viability compared with bFGF-loaded nanofibers or EGF surface-immobilized nanofibers alone. Layer-by-layer nanofibers A layer-by-layer (LBL) system has been applied to many types of biomedical devices, nanoparticles, films, and micelles (Krishnan et al. 2007; Hettiarachchi et al. 2009; Li et al. 2005; Galyean et al. 2009; Park et al. 2012). Many studies showed reduced initial burst with zero-order release kinetics using LBL devices (Poon et al. 2011; Shutava et al. 2009; Kim et al. 2008a; De Geest et al. 2006). The system was applied to electrospun nanofibers by stacking nanofiber sheets as shown in Fig. 2 (Kidoaki et al. 2005). This can control drug release by sheet barriers, similar to co-axial

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Fig. 3 Schematic diagram of hybrid system of the nanofiber with nano-and micro-sized devices and hydrogel based drug delivery system, and its release kinetics

nanofibers. There are several studies investigating LBL nanofibers using the stacking of sheets and deposition of coating materials to monolithic fibers (Yang et al. 2009; Nogueira et al. 2010; Lee et al. 2009). Multi-layer stacked nanofibers were introduced into the fabrication of multicell layers for three-dimensional tissue scaffolds, cell filtration, and drug delivery systems (Yang et al. 2009; Pham et al. 2006; Fathi-Azarbayjani and Chan 2010). Okuda et al. (2010) applied LBL nanofibers to time programmed dual-drug delivery system. The drug loading method was the co-electrospinning of a drug and polymer mixture. Sequential electrospinning of different solutions could fabricate a multilayered electrospun nanofiber mesh. Two kinds of poly(L-lactide-co-e-caploractone) (PLCL) solutions containing two different drugs were prepared and sequentially electrospun to fabricate nanofiber meshes with a time-programmed dual release function. PLCL polymer solution without drugs was also electrospun between the two different drug-loaded solution electrospinning steps to form a barrier for the purpose of time-controlled drug release. Therefore, the first drug-loaded layer was exposed on the top of the device and the second drug-loaded nanofiber sheet was sandwiched between the bottom and barrier layers. As the result, drugs loaded in first layer were released earlier than drugs loaded in second layer fabricated by the same drug-loading method of physical mixture co-electrospinning.

Hybrid system of nanofibers The electrospun nanofiber based drug delivery system have been hybridized with other type of devices, nano- and micro-sized carrier and hydrogel for efficient drug delivery and extended application of the mesh as shown in Fig. 3. Drug loading and release kinetics of the hybrid system and its advantages to the application was introduced in this section. Nanofiber with nano- and micro-sized devices For the improvement of drug-loading efficiency, release pattern, drug safety, and better functionality of the device, electrospun nanofibers have been hybridized with other type of devices. Nano- and micro-sized biomedical devices, such as polymeric nanoparticles, nanotubes, micelles, microspheres, and liposomes have been introduced into nanofibers for a controlled drug release system (Son and Yoo 2012; Liwen et al. 2008; Ionescu et al. 2010; Shao et al. 2011). Nano and micro-particulate drug embedded nanofibers are similar to co-axial nanofibers as they protect drug stability from organic solvents in the fabrication stage and prolong the period of drug delivery (Yoo et al. 2009). Moreover, it is an easier method than co-axial electrospinning because it uses a single nozzle. The development of multi-drug loading systems with nanofibers is more

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flexible with the hybridization of nanoparticles and nanofibers. Multi-drugs can be loaded into nanoparticles and nanofibers by co-electrospinning (Wang et al. 2010), and two different types of nanoparticles with different drugs can be electrospun with the polymer solution (Jo et al. 2009). Furthermore, drugs may not only be incorporated by co-electrospinning, but surface-immobilization is also possible in this system. In our group, micellized drugs were introduced on the surface of nanofibers by a surface-immobilized polymer chain (Chen et al. 2007; Son and Yoo 2012). A thermo-sensitive polymer, Pluronic F127, was used to modify the nanofiber surface of electrospun PCL-PEG and encapsulate the model drug, micellized dexamethasone. It showed that up to 98 % drugloading efficiency by the largest amount of Pluronic chain on the nanofiber and suppressed the release profile for 30 days. Even though surface immobilization is a safe method for drug stability, the amount of drugs being loaded into nanofibers is limited by the area of the nanofiber sheet. Nanofibers and hydrogels Hydrogels were introduced into nanofiber scaffolds because of their superior water retention, dual drug delivery or as a backbone device for the nanofiber in tissue engineering. The importance of extending the variety of nanofiber scaffold shapes was recognized and studies focused on micrometer scale nanofibrous hydrogels, which can be easily implanted inside the body (Lee et al. 2011, 2013a). PEG solution was cross-linked by UV on electrospun PCL nanofibers to obtain two different domains in the nanofiberbound hydrogel. For the proposed application of protein delivery, bovine serum albumin (BSA) was homogeneously loaded into nanofibers as a model protein. The BSAhybridized nanofibers localized in the hydrogel dramatically reduced the initial burst release because the hydrogel delayed the diffusion rate of BSA after released from the PCL nanofibers and the release was maintained for 30 days. Hydrogels have not only been combined with the nanofiber layer, but the hydrogel property has been directly introduced into electrospun nanofibers. Hydrogel-type materials are preferable over conventional hydrophobic materials in tissue engineering when considering cell adhesion and proliferation (Loh et al. 2010). Therefore, the hybridization of a hydrogel to a hydrophobic nanofiber improved the potency of the nanofiber in tissue engineering. The key point of hydrogel nanofibers is the electrospun polymer should maintain its fibrous structure in water with the hydrogel properties. However, hydrogel nanofibers exhibited high initial burst release of loaded drugs because of swelling. Surface treatment can reduce the initial burst of the nanofiber. PVA, which is a hydrophilic, biodegradable, and biocompatible polymer, was co-electrospun with

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drugs and surface-modified with fluorine to control the release period and rate (Im et al. 2010). According to the fluorine gas pressure treatment to the nanofiber surface, the drug release rate for 67 days was changed and even reduced the burst release during the initial stage because fluorination reduced the swelling property of PVA fibers in the aqueous phase. However, exposure of the nanofiber to extreme conditions, such as high temperature for fluorination, changes the morphology of nanofiber to a film, which would be not ideal for a biomolecule delivery device. Introducing hydrophilic chains into a hydrophobic polymer was performed and electrospun to produce a highly water-swellable nanofiber drug delivery system. Application Electrospun nanofiber as sustained drug delivery device has been mainly applied to tissue engineering. According to the purposes, the nanofiber can be a drug delivery carrier or a scaffold. As we explained in previous sections, incorporated drugs in/on nanofiber is not always released out. Surface immobilized proteins, peptides, other bioactive molecules are mainly related to cell adhesiveness and differentiation (Lee et al. 2013b; Choi et al. 2013). As a drug delivery device, mesh structure incorporating drugs have been targeted to antibacterial patch, mucosa, colon, wound site on skin, and cardiac tissue for local delivery system (Tian et al. 2012; Li et al. 2013; Yohe et al. 2012; Choi and Yoo 2010a). Patch type of drug loaded nanofiber is efficient device (Aduba et al. 2013). Electrospun nanofiber shows high mucoadhesive strength depending on composed polymers, and highly interacted with mucosal epithelial cells (Sharma et al. 2013). Also, sustained release of growth factors, DNAs, and siRNAs for a long period at wound site showed successful wound healing process (Kim and Yoo 2010, 2013a, b). As a scaffold of cells/tissue, ECM mimicking structure of the nanofibers is the main parameter to affect to cell differentiation. However, it was required to raise up cell attachment ratio to electrospun nanofiber composed of hydrophobic synthetic polymer such as PLGA, PCL, PLA, PU, and etc. that have advantages of proper mechanical strength and easy materials to be electrospun. Modification of the nanofiber with bioactive molecules surface can solve this problem. PLGA/PLGA-b-PEG-NH2 was prepared by electrospinning and a cell adhesive peptide, Arg–Gly–Asp (RGD) was immobilized on the surface for a potential wound dressing device (Kim and Park 2006). As the result, the surface property was changed to be hydrophilic, and NIH3T3 fibroblast successfully attached and proliferated a confluence state on RGD immobilized nanofiber for 5 days. Recently, the application of drug surface modified nanofiber was extended to stem cell differentiation;

Electrospun nanofibers for drug delivery systems

pluripotent stem cells, mesenchymal stem cell, and embryonic stem cell (Ren et al. 2013; Venugopal et al. 2012; Smith Callahan et al. 2013; Kolambkar et al. 2013). More interestingly, surface immobilized biomolecule technique has been improved to give gradients of incorporated drug on a nanofiber sheet to control stem cell differentiation (Zhang et al. 2012b; Zhu et al. 2012).

Conclusion The drug delivery system of electrospun nanofibers has been developed with synergic effects with the structural features of nanofibers. Therefore, various type of electrospun nanofiber were examined and grouped according to their drug-loading method. Most other literatures mainly distinguish electrospun nanofibers by co-electrospinning and chemical surface immobilization, which details direct and indirect drug incorporation into nanofibers and their hybridization system. According to the drug properties, such as hydrophobicity and hydrophilicity, bioactive molecules, and easy denaturation, we then differentiated the drug-loading abilities. With the development of many types of drug-loading methods, the initial burst release can be reduced, and zero-order release has also been exhibited (Immich et al. 2013; Zhang et al. 2006). Furthermore, hybridization with other types of biomedical devices elevated not only the drug release kinetics, but also produced scaffolds that can be applied as implantable tissue scaffold using hydrogel nanofibers. With many of advantages of nanofibers, such as flexible material selectivity, drugloading methods, variety of structural architectures and ECM mimicking structure, a massive variety of applications of nanofibers have been achieved, especially in tissue engineering. Released drugs from the nanofibers have been shown to control cellular metabolism, such as proliferation and differentiation (Choi and Yoo 2010a; Chen et al. 2012). They have also been shown to increase efficiency in local gene delivery systems. Furthermore, some meshes that have been modified with surface-immobilized proteins that are not released can be applied as cell sheets for attachment and differentiation. Therefore, it is expected that further applications of nanofibers with different drug combinations will be investigated and the advantages of the nanofibrous structural features will be exploited. Acknowledgments This work was supported by a grant from the National R&D Program for Cancer Control, Ministry for Health and Welfare, Republic of Korea.

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