Artificial Cells, Nanomedicine, and Biotechnology, 2014; Early Online: 1–6 Copyright © 2014 Informa Healthcare USA, Inc. ISSN: 2169-1401 print / 2169-141X online DOI: 10.3109/21691401.2014.965310
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Preparation and characterization of novel electrospun poly(e-caprolactone)-based nanofibrous scaffolds Alireza Valizadeh1,2,4, Mohsen Bakhtiary1,2, Abolfazl Akbarzadeh1,4, Roya Salehi4, Samad Mussa Frakhani1,2, Ommolbanin Ebrahimi1, Mohammad Rahmati-yamchi3 & Soodabeh Davaran1,4 1Department of Medical Nanotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz,
Iran, 2Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran, 3Department of Clinical Biochemistry, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran, and 4Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran tissue engineering (Mavis et al. 2009). A tissue engineering scaffold would be considered successful if it has biocompatibility, biodegradability, reproducibility, high porosity with interconnected pores, and no potential for serious immunological or foreign body reactions (Mavis et al. 2009). There are various methods for the preparation of nanoscaffolds, such as electrospinning, which has been widely used to produce nanoscale fiber mats of several biocompatible and biodegradable polymers, including synthetic, natural, or synthetic–natural polymer blends for tissue engineering purposes (Mavis et al. 2009, Thomas et al. 2006, Zhang et al. 2005, Sarasam and Madihally 2005). Fibers with diameters less than 1000 nm (or 1 mm) are defined as nanofibers, and are characterized by their long length, small diameter and high surface area per unit volume (Valizadeh and Farkhani 2013). Nanofibers (NFs) can be produced by several processing techniques, such as drawing, template synthesis, phase separation, self-assembly, electrospinning and so on (Valizadeh and Farkhani 2013, Kim 2006, Ondarcuhu and Joachim 2007, Ratanajiajaroen and Ohshima 2012, Liu et al. 2012, Whitesides and Grzybowski 2002). A comparison of all the techniques mentioned reveals that electrospinning (ES) is the only method that can be further developed for mass production of individual, continuous nanofibers from various polymers. Moreover, ES is a simple technique which produces one-dimensional (1D) continuous fibrous structures, and is cost-effective, versatile, scalable and reliable (Valizadeh and Farkhani 2013, Huang et al. 2003). ES allows the fabrication of polymer fibers with diameters varying from 3nm to greater than 5 mm and gives us the capability to design and the operating parameters for synthesis of interesting novel nanofibers which can be used to fabricate nanofiber scaffolds for tissue engineering. Electrospinning is a promising technique for the preparation of micro- and nanofibers of polyesters like poly (e-caprolactone) (PCL),
Abstract Nanofibrous scaffolds have many advantages that make them excellent candidates for tissue engineering applications. The scaffolds with high surface area to volume ratio favor cell adhesion, proliferation, migration and differentiation. In the present study, the preparation of electrospun poly (e-caprolactone)polyethylene glycol-poly (e-caprolactone) (PCL-PEG-PCL) nanofibers is shown for the first time. PCL-PEG-PCL copolymers were synthesized using a ring-opening polymerization method. The polymers were characterized by FT-IR, 1H NMR and DSC. Nanofibers with mean diameters ranging from 60 to 170 nm were obtained by the electrospinning method. Their morphology was evaluated by scanning electron microscopy (SEM). An MTT assay was used to compare the number of cells in the nanofiber scaffold. It was found that the morphology and diameter of the nanofiber depended on the chemical composition and molecular weight of the PEG segment of the copolymer used for electrospinning. Increasing the molecular weight of PEG blocks from 2000 to 6000 led to a decrease of the diameter of the fibers and the formation of beads. Keywords: electrospinning, MTT assay, nanofibrous scaffolds, PCL-PEG-PCL
Introduction Polymeric biomaterials play a crucial role in tissue engineering by serving as synthetic frameworks, commonly referred to as scaffolds, for cell attachment, proliferation and growth, and ultimately leading to new tissue formation. Both synthetic polymers and natural polymers have been extensively investigated as biodegradable polymeric biomaterials in tissue engineering. Controlling and regulating the potential of natural tissue regeneration mechanisms and the use of mimics of the natural extracellular matrix (ECM) for defect repair or even organ regeneration is the challenge in
Correspondence: Prof. Soodabeh Davaran, Department of Medical Nanotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz 51664, Iran. E-mail: [email protected] (Received 2 September 2014; revised 9 September 2014; accepted 10 September 2014)
1
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2 A. Valizadeh et al. polylactide, polyglycolide (PLGA) and other biodegradable polyesters. Polycaprolactone (PCL) is very attractive in tissue engineering because of its good biocompatibility and processability (Hutmacher 2001, Hutmacher et al. 2001, Huang et al. 2006). The applications of PCL might be limited because its degradation and resorption kinetics are considerably slower than other aliphatic polyesters. Also, due to its hydrophobic character and high crystallinity and elasticity, PCL is currently under study for use as a potential material for bone regeneration (Hamlekhan et al. 2011). The hydrophobicity and crystallinity of the PCL can be increased by preparation of its copolymers with hydrophilic monomers or polymers. PEGs have been most commonly copolymerized with CL to prepare highly biocompatible, hydrophilic, non-toxic, non-immunogenic and non-antigenic copolymers. Such properties reduce protein adsorption and enhance resistance to bacterial and animal cell adhesion. Modifying conventional polyesters with polyethylene glycol can improve certain polyester characteristics, yet can adversely affect others. Accordingly, this work shows a method for preparation of a PCL-PEG-PCL triblock copolymer in a way that retains the favorable properties of polyethylene glycol, while retaining the advantages of PCL. This facilitates the spinning of the PEG-modified PCL using conventional spinning equipment. In this study, the following three factors and their role in fiber size and morphology were evaluated: (i) polymer composition, (ii) PEG molecular weight (Mw), and (iii) concentration of polymer solution. Triblock PCL-PEG-PCL copolymers were used in an organic (hydrophobic) solvent. These polymers
were chosen because they have been used and characterized extensively for biomedical applications.
Materials and methods Materials
e-Caprolactone (e-CL), polyethylene glycol (PEG) with Mws of 2000, 3000, 4000, and 6000, chloroform and methanol were purchased from Merck Chemical Co. Stannous octoate Sn(Oct)2 was supplied by Alfa Aesar.
Polymer synthesis Synthesis of PCL polymer PCL homopolymer was prepared by ring-opening polymerization of e-CL using stannous octoate as a catalyst (Sn(Oct)2). Briefly, 10.0 g of e-CL and 0.05 g (0.5 wt %) of Sn (Oct)2 were added to a 25 ml round-bottom flask. The suspension was heated to 120 ˚C under stirring in a nitrogen atmosphere for 8 h. The resulting polymer was dissolved in methylene chloride and precipitated in an excess of cold hexane, and it was dried at room temperature for 48 h.
Synthesis of PCL/PEG/PCL triblock copolymer PCL/PEG/PCL copolymers with four different compositions were prepared by ring-opening polymerization of e-CL in the presence of PEG, using stannous octoate as catalyst. Briefly, 10 g of e-CL, 1 g of PEG with different Mws (PEG2000, PEG3000, PEG4000, or PEG6000), and Sn(Oct)2 (0.5 wt% of monomers) were added to a 25 ml round-bottom flask in a nitrogen atmosphere. The suspension was heated to 120˚C under stirring and in a nitrogen atmosphere for 8 h. The resulting polymer was dissolved in methylene chloride and precipitated in an excess of cold hexane and dried at room temperature for 48 h.
Figure 1. PCL-PEG4000-PCL triblock copolymers.
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Novel electrospun poly(e-caprolactone)-based nanofibrous scaffolds 3
Figure 2. 1H-NMR of PCL-PEG4000-PCL triblock copolymers.
Characterization of chemical structure of the polymer Characterization of the chemical structures of polymers was carried out by FT-IR and 1H NMR. The FT-IR spectra of polymers were obtained by using a Shimadzu 8400s Infrared Spectrophotometer (Kyoto, Japan). 1H-NMR spectra of polymers were obtained by using Bruker Avance 400 instruments. CDCl3/CD3OD and tetramethylsilane (TMS) were used as the solvent and internal reference standard, respectively.
Thermal analysis of polymers Two mg of copolymer samples was loaded in aluminum pans along with the standard reference aluminum in the DSC (Jade, PerkinElemer, USA). The DSC was recorded between 35°C and 250°C at a scan rate of 10°C/min.
Preparation of polymeric nanofibrous scaffolds by the electrospinning method PCL or PCL/PEG/PCL was dissolved in chloroform: methanol (3:1 v/v ratio) to produce a solution of 10%- 40% w/v concentration. The solutions were magnetically stirred at room temperature for 8 h at 25°C. The solution was placed in a 10 ml plastic syringe with a blunt-ended metal needle tip (gauge 16). The spinning solution was delivered at a controlled feed rate of 3ml/h, and the voltage between the needle tip and the
ground collector on the electrospinning device (Fanavaran Nano-Meghyas, Iran) was at a range of 12–25 KV. The distance from the nozzle to the collector and the rotating speed of the drum were 120 mm and 1000 rpm, respectively. The metal collector was covered with an aluminum foil. All electrospinning experiments were carried out at room temperature (Salehi et al. 2013, Wang et al. 2012, Jeong et al. 2010, Koev et al. 2010).
Morphological characterization of nanofibrous scaffolds The morphology of the electrospun fibers was observed by scanning electron microscopy (SEM). For the performance of the SEM analyses, the mats were vacuum-coated with gold and analyzed using SEM (KYKY-EM3200, China) at 25 KV. For each image, at least 13 different fiber segments were randomly selected and their diameters were measured to generate an average fiber diameter.
Results and discussion Chemical structure of PCL polymer and PCL/PEG/PCL copolymers The chemical structure of PCL-PEG-PCL copolymers was studied by FT-IR and 1H-NMR spectroscopy. Typical spectra for PCL-PEG6000-PEG are given in Figures 1 and 2.
Table I. Polymerization of e-CL with different Mw values of PEG (wt% 10%), and characterization of copolymers. Sample PCL PCL-PEG2000-PCL PCL-PEG3000-PCL PCL-PEG4000-PCL PCL-PEG6000-PCL
Mw of PEG
PEG (wt. %)
– 2000 3000 4000 6000
0 10 10 10 10
Mw ( 10 4) 13.5 7.6 8.1 8.4 9.2
Mn ( 10 4)
Mw/Mn
11.25 5.7 7.29 6 7
1.2 1.33 1.11 1.4 1.3
umber average molecular weight (Mn) and Weight average molecular weight (Mw) measured by GPC (calibrated with N polystyrene standards).
4 A. Valizadeh et al. Table II. The glass transition and melting temperatures of copolymers. Sample Tg of copolymer (˚C) Tm of copolymer (˚C)
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PCL-PEG2000-PCL PCL-PEG3000-PCL PCL-PEG4000-PCL PCL-PEG6000-PCL
57.5 58.4 59.6 60.5
33.4 31.6 30.34 29.4
Figure 1 shows FT-IR spectra of PCL-PEG4000-PCL triblock copolymers. Stretching vibrations of the ester carbonyl group appear at 1736 cm 1. The absorption bands at 1250–1100 cm 1 are attributed to the C–O–C stretching vibrations of the repeated –OCH2CH2 units of PEG. The absorption band at 3442 cm 1 is assigned to terminal –OH groups in the copolymer. All the C–H stretching bonds are centered at 2980–2800 cm 1. The FT-IR spectra of other PCL-PEG-PCL copolymers were similar to those of PCL-PEG4000-PCL. The 1H-NMR spectra also confirmed the structure of PCLPEG4000-PCL triblock copolymers (Figure 2). The strong signals of the a, b, g, d, methylene protons to the carbonyl group of PCL were detected at 1.26, 1.53, 2.20, and 3.52 ppm. The sharp peak at 3.28 ppm is attributed to the methylene protons of ─CH2CH2O─ in PEG units in the block copolymer. The very weak peaks at 3.52 ppm are attributed to the methylene protons of ─O─CH2─CH2─ in a PEG end unit that linked with PCL blocks. The Mws of the polymers were determined by gel permeation chromatography (GPC). The results are given in Table I. It can be seen from Table I that the weight average Mw of PEG in the feed has a significant effect on the Mw of the resulting triblock copolymer. Similarly, it was found that the Mw values of PCL-PEG-PCL copolymers increased with the increase of the Mw values of PEG from 2000 to 6000.
Thermal properties of polymers For studying the dependence of the thermal behavior of the copolymers on their composition, DSC measurements were used. The glass transition (Tg), and the melting temperatures (Tm) of PCL-PEG-PCL copolymers are listed in Table II. The Tg values of all copolymers are shifted slightly to higher temperature in comparison to the Tg value of PCL homopolymer ( 63˚C). Furthermore, Tg values tend to decrease with increasing the Mw of PEG units. Peak melting temperatures for PCL units in copolymers were between 56˚C and 63˚C.
Morphology of nanofibrous scaffolds The morphology of the fibers was investigated using SEM. The morphology and size distributions of the PCL nanofibers at 15 KX and 7.5 KX resolution are shown in Figure 3. The PCL fibers exhibited smooth surfaces and uniform structures without any beads. As shown in the Figure 3, nanofibers prepared from PCL showed good properties in terms of diameter, uniformity and non-beaded area. In this study, four different compositions of PCL-PEGPCL polymer (PCL-PEG2000-PCL, PCL-PEG3000-PCL, PCLPEG4000-PCL, and PCL-PEG6000-PCL) in dichloromethane solvent were investigated after pre-modification by gold coating. Three different concentrations of PCL-PEG-PCL triblock copolymer solutions (10 wt%, 20 wt% and 30 wt %) were used. The SEM images of the expected nanofibers are given in Figure 4. It was observed that at lower concentration (below 30%), the polymer jet was not stable and fibers were found to be beaded. Applied voltages ranging from 12 to 25 kV were tested at the ambient relative humidity of approximately 35%, and it was found that a range of 23–25kV was optimal for elec-
Figure 3. SEM images of the PCL nanofibers; (A) 15KX resolution of PCL, (B) Invert color effected figure for better view of scaffold depth, (C) 7.5KX resolution of PCL, (D) Invert color effected for better view of scaffold depth.
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Novel electrospun poly(e-caprolactone)-based nanofibrous scaffolds 5
Figure 4. SEM images of (horizontal setup, 10 cm, flow rate of 3 ml/h) electrospun nanofibers, 25 kV, 30% wt polymer, 30 KX: (A); PCL-PEG2000-PCL, (B); PCL-PEG3000-PCL, (C); PCL-PEG4000-PCL, (D);PCL-PEG6000-PCL.
trospinning of PCL-PEG-PCL fibers (Figure 4). At voltages below 25kV, an unsteady Taylor cone was observed through discontinuous electrospinning of PCL-PEG-PCL fibers. It was observed that at lower voltage (12–20 kV), the fibers tended to be thicker and formed beads. At a polymer concentration of 30% and applied voltage of 25 kV, the diameter of nanofibers was dependent on the Mw of the PEG central segment of the copolymer. As shown in Figure 4, when other parameters are kept constant and the Mw of PEG is increased, the nanofiber diameter decreases; however, the bead formation and bead sizes increase. The lowest average fiber diameter (35.5 nm) was obtained using PCL-PEG6000-PCL. The beaded structure of PCL-PEG-PCL nanofibers shows that a stable Taylor cone could not be obtained. The bead formation was eliminated at low Mw of PEG (PCL-PEG2000-PCL).
Conclusion In this study, a series of PCL-PEG-PCL block copolymers were synthesized by ring opening polymerization method and used for the preparation of biodegradable nanofibers. With the increasing Mw of PEG, the fiber diameter decreased and the fiber morphology became finer. The results shown in the present study are of significant importance for the design of novel, nanostructured fibrous biomaterials for drug delivery and tissue engineering.
Authors’ contributions SD conceived the study and participated in its design and coordination. AV participated in the sequence alignment and drafted the manuscript. AA, OE, SF, and MB helped in drafting the manuscript. All authors read and approved the final manuscript.
Declaration of interest The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper. The authors are grateful for the financial support from: 1. The Department of Medical Nanotechnology, Faculty of Advanced Medical Sciences, Iran, national Science Foundation Tabriz, Iran. 2. The Research Center for Pharmaceutical Nanotechnology (RCPN), Tabriz University of Medical Sciences, Tabriz-IRAN.
References Hamlekhan A, Moztarzadeh F, Mozafari M, Azami M, Nezafati N. 2011. Preparation of laminated poly (e-caprolactone)-gelatinhydroxyapatite nanocomposite scaffold bioengineered via compound techniques for bone substitution. Biomatter. 1:91–101. Huang MH, Li S, Hutmacher DW, Coudane J, Vert M. 2006. Degradation characteristics of poly (ε-caprolactone)-based copolymers and blends. J Appl Polym Sci. 102:1681–1687. Huang ZM, Zhang YZ, Kotaki M, Ramakrishna S. 2003. A review on polymer nanofibers by electrospinning and their applications in nanocomposites. Compos Sci Technol. 63:2223–2253. Hutmacher DW, Schantz T, Zein I, Ng KW, Teoh SH, Tan KC. 2001. Mechanical properties and cell cultural response of polycaprolactone scaffolds designed and fabricated via fused deposition modeling. J Biomed Mater Res. 55:203–216. Hutmacher DW. 2001. Scaffold design and fabrication technologies for engineering tissues—state of the art and future perspectives. J Biomater Sci Polym Ed. 12:107–124. Jeong GS, Chung S, Kim CB, Lee SH. 2010. Applications of micromixing technology. Analyst. 135:460–473. Kim HY. 2006. Method of manufacturing a continuous filament by electrospinning and continuous filament manufactured thereby. WO Patent 2,006,135,147. Koev ST, Dykstra PH, Luo X, Rubloff GW, Bentley WE, Payne GF, Ghodssi R. 2010. Chitosan: an integrative biomaterial for lab-on-achip devices. Lab Chip. 10:3026–3042.
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6 A. Valizadeh et al. Liu M, Cheng Z, Yan J, Qiang L, Ru X, Liu F, et al. 2012. Preparation and characterization of TiO2 nanofibers via using polylactic acid as template. J Appl Polym Sci. Mavis B, Demirtas¸ TT, Gümüs¸dereliog˘lu M, Gündüz G, Colak U. 2009. Synthesis, characterization and osteoblastic activity of polycaprolactone nanofibers coated with biomimetic calcium phosphate. Acta Biomate. 5:3098–3111. Ondarcuhu T, Joachim C. 2007. Drawing a single nanofibre over hundreds of microns. Europhys Lett. 42:215. Ratanajiajaroen P, Ohshima M. 2012. Preparation of highly porous b-chitin structure through nonsolvent-solvent exchange-induced phase separation and supercritical CO2 drying. J Supercrit Fluids. Salehi R, Iranib M, Rashidic MR, Aroujalian A, Raisi A, Eskandani M, et al. 2013. Stimuli-responsive nanofibers prepared from poly (N-isopropylacrylamide-acrylamide-vinylpyrrolidone) by electrospinning as an anticancer drug delivery. Des Monomers Polym. 16:515–527.
Sarasam A, Madihally SV. 2005. Characterization of chitosan– polycaprolactone blends for tissue engineering applications. Biomaterials. 26:5500–5508. Thomas V, Dean DR, Vohra YK. 2006. Nanostructured biomaterials for regenerative medicine. Curr Nanosci.2:155–177. Valizadeh A, Farkhani SM. 2013. Electrospinning and electrospun nanofibres. IET Nanobiotechnol. Wang H, Yakai F, Zichen F, Wenjie Y, Musammir K. 2012. Co-electrospun blends of PU and PEG as potential biocompatible scaffolds for small-diameter vascular tissue engineering. Mater Sci Eng C. 32:2306–2315. Whitesides GM, Grzybowski B. 2002. Self-assembly at all scales. Science. 295:2418–2421. Zhang Y, Ouyang H, Lim CT, Ramakrishna S, Huang ZM. 2005. Electrospinning of gelatin fibers and gelatin/PCL composite fibrous scaffolds. J Biomed Mater Res B Appl Biomater. 72: 156–165.
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Preparation and characterization of novel electrospun poly(e-caprolactone)-based nanofibrous scaffolds Alireza Valizadeh1,2,4, Mohsen Bakhtiary1,2, Abolfazl Akbarzadeh1,4, Roya Salehi4, Samad Mussa Frakhani1,2, Ommolbanin Ebrahimi1, Mohammad Rahmati-yamchi3 & Soodabeh Davaran1,4 1Department of Medical Nanotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz,
Iran, 2Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran, 3Department of Clinical Biochemistry, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran, and 4Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran tissue engineering (Mavis et al. 2009). A tissue engineering scaffold would be considered successful if it has biocompatibility, biodegradability, reproducibility, high porosity with interconnected pores, and no potential for serious immunological or foreign body reactions (Mavis et al. 2009). There are various methods for the preparation of nanoscaffolds, such as electrospinning, which has been widely used to produce nanoscale fiber mats of several biocompatible and biodegradable polymers, including synthetic, natural, or synthetic–natural polymer blends for tissue engineering purposes (Mavis et al. 2009, Thomas et al. 2006, Zhang et al. 2005, Sarasam and Madihally 2005). Fibers with diameters less than 1000 nm (or 1 mm) are defined as nanofibers, and are characterized by their long length, small diameter and high surface area per unit volume (Valizadeh and Farkhani 2013). Nanofibers (NFs) can be produced by several processing techniques, such as drawing, template synthesis, phase separation, self-assembly, electrospinning and so on (Valizadeh and Farkhani 2013, Kim 2006, Ondarcuhu and Joachim 2007, Ratanajiajaroen and Ohshima 2012, Liu et al. 2012, Whitesides and Grzybowski 2002). A comparison of all the techniques mentioned reveals that electrospinning (ES) is the only method that can be further developed for mass production of individual, continuous nanofibers from various polymers. Moreover, ES is a simple technique which produces one-dimensional (1D) continuous fibrous structures, and is cost-effective, versatile, scalable and reliable (Valizadeh and Farkhani 2013, Huang et al. 2003). ES allows the fabrication of polymer fibers with diameters varying from 3nm to greater than 5 mm and gives us the capability to design and the operating parameters for synthesis of interesting novel nanofibers which can be used to fabricate nanofiber scaffolds for tissue engineering. Electrospinning is a promising technique for the preparation of micro- and nanofibers of polyesters like poly (e-caprolactone) (PCL),
Abstract Nanofibrous scaffolds have many advantages that make them excellent candidates for tissue engineering applications. The scaffolds with high surface area to volume ratio favor cell adhesion, proliferation, migration and differentiation. In the present study, the preparation of electrospun poly (e-caprolactone)polyethylene glycol-poly (e-caprolactone) (PCL-PEG-PCL) nanofibers is shown for the first time. PCL-PEG-PCL copolymers were synthesized using a ring-opening polymerization method. The polymers were characterized by FT-IR, 1H NMR and DSC. Nanofibers with mean diameters ranging from 60 to 170 nm were obtained by the electrospinning method. Their morphology was evaluated by scanning electron microscopy (SEM). An MTT assay was used to compare the number of cells in the nanofiber scaffold. It was found that the morphology and diameter of the nanofiber depended on the chemical composition and molecular weight of the PEG segment of the copolymer used for electrospinning. Increasing the molecular weight of PEG blocks from 2000 to 6000 led to a decrease of the diameter of the fibers and the formation of beads. Keywords: electrospinning, MTT assay, nanofibrous scaffolds, PCL-PEG-PCL
Introduction Polymeric biomaterials play a crucial role in tissue engineering by serving as synthetic frameworks, commonly referred to as scaffolds, for cell attachment, proliferation and growth, and ultimately leading to new tissue formation. Both synthetic polymers and natural polymers have been extensively investigated as biodegradable polymeric biomaterials in tissue engineering. Controlling and regulating the potential of natural tissue regeneration mechanisms and the use of mimics of the natural extracellular matrix (ECM) for defect repair or even organ regeneration is the challenge in
Correspondence: Prof. Soodabeh Davaran, Department of Medical Nanotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz 51664, Iran. E-mail: [email protected] (Received 2 September 2014; revised 9 September 2014; accepted 10 September 2014)
1
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2 A. Valizadeh et al. polylactide, polyglycolide (PLGA) and other biodegradable polyesters. Polycaprolactone (PCL) is very attractive in tissue engineering because of its good biocompatibility and processability (Hutmacher 2001, Hutmacher et al. 2001, Huang et al. 2006). The applications of PCL might be limited because its degradation and resorption kinetics are considerably slower than other aliphatic polyesters. Also, due to its hydrophobic character and high crystallinity and elasticity, PCL is currently under study for use as a potential material for bone regeneration (Hamlekhan et al. 2011). The hydrophobicity and crystallinity of the PCL can be increased by preparation of its copolymers with hydrophilic monomers or polymers. PEGs have been most commonly copolymerized with CL to prepare highly biocompatible, hydrophilic, non-toxic, non-immunogenic and non-antigenic copolymers. Such properties reduce protein adsorption and enhance resistance to bacterial and animal cell adhesion. Modifying conventional polyesters with polyethylene glycol can improve certain polyester characteristics, yet can adversely affect others. Accordingly, this work shows a method for preparation of a PCL-PEG-PCL triblock copolymer in a way that retains the favorable properties of polyethylene glycol, while retaining the advantages of PCL. This facilitates the spinning of the PEG-modified PCL using conventional spinning equipment. In this study, the following three factors and their role in fiber size and morphology were evaluated: (i) polymer composition, (ii) PEG molecular weight (Mw), and (iii) concentration of polymer solution. Triblock PCL-PEG-PCL copolymers were used in an organic (hydrophobic) solvent. These polymers
were chosen because they have been used and characterized extensively for biomedical applications.
Materials and methods Materials
e-Caprolactone (e-CL), polyethylene glycol (PEG) with Mws of 2000, 3000, 4000, and 6000, chloroform and methanol were purchased from Merck Chemical Co. Stannous octoate Sn(Oct)2 was supplied by Alfa Aesar.
Polymer synthesis Synthesis of PCL polymer PCL homopolymer was prepared by ring-opening polymerization of e-CL using stannous octoate as a catalyst (Sn(Oct)2). Briefly, 10.0 g of e-CL and 0.05 g (0.5 wt %) of Sn (Oct)2 were added to a 25 ml round-bottom flask. The suspension was heated to 120 ˚C under stirring in a nitrogen atmosphere for 8 h. The resulting polymer was dissolved in methylene chloride and precipitated in an excess of cold hexane, and it was dried at room temperature for 48 h.
Synthesis of PCL/PEG/PCL triblock copolymer PCL/PEG/PCL copolymers with four different compositions were prepared by ring-opening polymerization of e-CL in the presence of PEG, using stannous octoate as catalyst. Briefly, 10 g of e-CL, 1 g of PEG with different Mws (PEG2000, PEG3000, PEG4000, or PEG6000), and Sn(Oct)2 (0.5 wt% of monomers) were added to a 25 ml round-bottom flask in a nitrogen atmosphere. The suspension was heated to 120˚C under stirring and in a nitrogen atmosphere for 8 h. The resulting polymer was dissolved in methylene chloride and precipitated in an excess of cold hexane and dried at room temperature for 48 h.
Figure 1. PCL-PEG4000-PCL triblock copolymers.
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Novel electrospun poly(e-caprolactone)-based nanofibrous scaffolds 3
Figure 2. 1H-NMR of PCL-PEG4000-PCL triblock copolymers.
Characterization of chemical structure of the polymer Characterization of the chemical structures of polymers was carried out by FT-IR and 1H NMR. The FT-IR spectra of polymers were obtained by using a Shimadzu 8400s Infrared Spectrophotometer (Kyoto, Japan). 1H-NMR spectra of polymers were obtained by using Bruker Avance 400 instruments. CDCl3/CD3OD and tetramethylsilane (TMS) were used as the solvent and internal reference standard, respectively.
Thermal analysis of polymers Two mg of copolymer samples was loaded in aluminum pans along with the standard reference aluminum in the DSC (Jade, PerkinElemer, USA). The DSC was recorded between 35°C and 250°C at a scan rate of 10°C/min.
Preparation of polymeric nanofibrous scaffolds by the electrospinning method PCL or PCL/PEG/PCL was dissolved in chloroform: methanol (3:1 v/v ratio) to produce a solution of 10%- 40% w/v concentration. The solutions were magnetically stirred at room temperature for 8 h at 25°C. The solution was placed in a 10 ml plastic syringe with a blunt-ended metal needle tip (gauge 16). The spinning solution was delivered at a controlled feed rate of 3ml/h, and the voltage between the needle tip and the
ground collector on the electrospinning device (Fanavaran Nano-Meghyas, Iran) was at a range of 12–25 KV. The distance from the nozzle to the collector and the rotating speed of the drum were 120 mm and 1000 rpm, respectively. The metal collector was covered with an aluminum foil. All electrospinning experiments were carried out at room temperature (Salehi et al. 2013, Wang et al. 2012, Jeong et al. 2010, Koev et al. 2010).
Morphological characterization of nanofibrous scaffolds The morphology of the electrospun fibers was observed by scanning electron microscopy (SEM). For the performance of the SEM analyses, the mats were vacuum-coated with gold and analyzed using SEM (KYKY-EM3200, China) at 25 KV. For each image, at least 13 different fiber segments were randomly selected and their diameters were measured to generate an average fiber diameter.
Results and discussion Chemical structure of PCL polymer and PCL/PEG/PCL copolymers The chemical structure of PCL-PEG-PCL copolymers was studied by FT-IR and 1H-NMR spectroscopy. Typical spectra for PCL-PEG6000-PEG are given in Figures 1 and 2.
Table I. Polymerization of e-CL with different Mw values of PEG (wt% 10%), and characterization of copolymers. Sample PCL PCL-PEG2000-PCL PCL-PEG3000-PCL PCL-PEG4000-PCL PCL-PEG6000-PCL
Mw of PEG
PEG (wt. %)
– 2000 3000 4000 6000
0 10 10 10 10
Mw ( 10 4) 13.5 7.6 8.1 8.4 9.2
Mn ( 10 4)
Mw/Mn
11.25 5.7 7.29 6 7
1.2 1.33 1.11 1.4 1.3
umber average molecular weight (Mn) and Weight average molecular weight (Mw) measured by GPC (calibrated with N polystyrene standards).
4 A. Valizadeh et al. Table II. The glass transition and melting temperatures of copolymers. Sample Tg of copolymer (˚C) Tm of copolymer (˚C)
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PCL-PEG2000-PCL PCL-PEG3000-PCL PCL-PEG4000-PCL PCL-PEG6000-PCL
57.5 58.4 59.6 60.5
33.4 31.6 30.34 29.4
Figure 1 shows FT-IR spectra of PCL-PEG4000-PCL triblock copolymers. Stretching vibrations of the ester carbonyl group appear at 1736 cm 1. The absorption bands at 1250–1100 cm 1 are attributed to the C–O–C stretching vibrations of the repeated –OCH2CH2 units of PEG. The absorption band at 3442 cm 1 is assigned to terminal –OH groups in the copolymer. All the C–H stretching bonds are centered at 2980–2800 cm 1. The FT-IR spectra of other PCL-PEG-PCL copolymers were similar to those of PCL-PEG4000-PCL. The 1H-NMR spectra also confirmed the structure of PCLPEG4000-PCL triblock copolymers (Figure 2). The strong signals of the a, b, g, d, methylene protons to the carbonyl group of PCL were detected at 1.26, 1.53, 2.20, and 3.52 ppm. The sharp peak at 3.28 ppm is attributed to the methylene protons of ─CH2CH2O─ in PEG units in the block copolymer. The very weak peaks at 3.52 ppm are attributed to the methylene protons of ─O─CH2─CH2─ in a PEG end unit that linked with PCL blocks. The Mws of the polymers were determined by gel permeation chromatography (GPC). The results are given in Table I. It can be seen from Table I that the weight average Mw of PEG in the feed has a significant effect on the Mw of the resulting triblock copolymer. Similarly, it was found that the Mw values of PCL-PEG-PCL copolymers increased with the increase of the Mw values of PEG from 2000 to 6000.
Thermal properties of polymers For studying the dependence of the thermal behavior of the copolymers on their composition, DSC measurements were used. The glass transition (Tg), and the melting temperatures (Tm) of PCL-PEG-PCL copolymers are listed in Table II. The Tg values of all copolymers are shifted slightly to higher temperature in comparison to the Tg value of PCL homopolymer ( 63˚C). Furthermore, Tg values tend to decrease with increasing the Mw of PEG units. Peak melting temperatures for PCL units in copolymers were between 56˚C and 63˚C.
Morphology of nanofibrous scaffolds The morphology of the fibers was investigated using SEM. The morphology and size distributions of the PCL nanofibers at 15 KX and 7.5 KX resolution are shown in Figure 3. The PCL fibers exhibited smooth surfaces and uniform structures without any beads. As shown in the Figure 3, nanofibers prepared from PCL showed good properties in terms of diameter, uniformity and non-beaded area. In this study, four different compositions of PCL-PEGPCL polymer (PCL-PEG2000-PCL, PCL-PEG3000-PCL, PCLPEG4000-PCL, and PCL-PEG6000-PCL) in dichloromethane solvent were investigated after pre-modification by gold coating. Three different concentrations of PCL-PEG-PCL triblock copolymer solutions (10 wt%, 20 wt% and 30 wt %) were used. The SEM images of the expected nanofibers are given in Figure 4. It was observed that at lower concentration (below 30%), the polymer jet was not stable and fibers were found to be beaded. Applied voltages ranging from 12 to 25 kV were tested at the ambient relative humidity of approximately 35%, and it was found that a range of 23–25kV was optimal for elec-
Figure 3. SEM images of the PCL nanofibers; (A) 15KX resolution of PCL, (B) Invert color effected figure for better view of scaffold depth, (C) 7.5KX resolution of PCL, (D) Invert color effected for better view of scaffold depth.
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Novel electrospun poly(e-caprolactone)-based nanofibrous scaffolds 5
Figure 4. SEM images of (horizontal setup, 10 cm, flow rate of 3 ml/h) electrospun nanofibers, 25 kV, 30% wt polymer, 30 KX: (A); PCL-PEG2000-PCL, (B); PCL-PEG3000-PCL, (C); PCL-PEG4000-PCL, (D);PCL-PEG6000-PCL.
trospinning of PCL-PEG-PCL fibers (Figure 4). At voltages below 25kV, an unsteady Taylor cone was observed through discontinuous electrospinning of PCL-PEG-PCL fibers. It was observed that at lower voltage (12–20 kV), the fibers tended to be thicker and formed beads. At a polymer concentration of 30% and applied voltage of 25 kV, the diameter of nanofibers was dependent on the Mw of the PEG central segment of the copolymer. As shown in Figure 4, when other parameters are kept constant and the Mw of PEG is increased, the nanofiber diameter decreases; however, the bead formation and bead sizes increase. The lowest average fiber diameter (35.5 nm) was obtained using PCL-PEG6000-PCL. The beaded structure of PCL-PEG-PCL nanofibers shows that a stable Taylor cone could not be obtained. The bead formation was eliminated at low Mw of PEG (PCL-PEG2000-PCL).
Conclusion In this study, a series of PCL-PEG-PCL block copolymers were synthesized by ring opening polymerization method and used for the preparation of biodegradable nanofibers. With the increasing Mw of PEG, the fiber diameter decreased and the fiber morphology became finer. The results shown in the present study are of significant importance for the design of novel, nanostructured fibrous biomaterials for drug delivery and tissue engineering.
Authors’ contributions SD conceived the study and participated in its design and coordination. AV participated in the sequence alignment and drafted the manuscript. AA, OE, SF, and MB helped in drafting the manuscript. All authors read and approved the final manuscript.
Declaration of interest The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper. The authors are grateful for the financial support from: 1. The Department of Medical Nanotechnology, Faculty of Advanced Medical Sciences, Iran, national Science Foundation Tabriz, Iran. 2. The Research Center for Pharmaceutical Nanotechnology (RCPN), Tabriz University of Medical Sciences, Tabriz-IRAN.
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