Highly Porous Core–Shell Polymeric Fiber Network - American

Jul 7, 2011 - Highly Porous CoreАShell Polymeric Fiber Network. Muhammad Gulfam,. † ... core and polymer shell have been generated by the electrosp...
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Highly Porous CoreShell Polymeric Fiber Network Muhammad Gulfam,† Jong Min Lee,† Ji-eun Kim,† Dong Woo Lim,† Eun Kyu Lee,‡ and Bong Geun Chung*,† † ‡

Department of Bionano Engineering, Hanyang University, Ansan, 426-791 Korea College of Bionanotechnology, Kyungwon University, Seongnam, 461-701 Korea ABSTRACT: Coreshell nanofibers are of great interest in the field of tissue engineering and cell biology. We fabricated porous coreshell fiber networks using an electrospinning system with a water-immersed collector. We hypothesized that the phase separation and solvent evaporation process would enable the control of the pore formation on the coreshell fiber networks. To synthesize porous coreshell fiber networks, we used polycaprolactone (PCL) and gelatin. Quantitative analysis showed that the sizes of gelatinPCL coreshell nanofibers increased with PCL concentrations. We also observed that the shapes of the pores created on the PCL fiber networks were elongated, whereas the gelatinPCL coreshell fiber networks had circular pores. The surface areas of porous nanofibers were larger than those of the nonporous nanofibers due to the highly volatile solvent and phase separation process. The porous coreshell fiber network was also used as a matrix to culture various cell types, such as embryonic stem cells, breast cancer cells, and fibroblast cells. Therefore, this porous coreshell polymeric fiber network could be a potentially powerful tool for tissue engineering and biological applications.

1. INTRODUCTION The electrospinning technique that can create nanofibers is of great interest in the field of tissue engineering applications.15 The electrospun nanofibers have a number of advantages, such as high surface-to-volume ratio and tunable porosity.69 Given these properties, nanofibers have been widely used for various applications, such as filtration, scaffold,1014 drug delivery,1517 conductive nanowire,18 and bionanosensor.19 Although core shell nanofibers are synthesized using the coaxial electrospinning technique, the porosity of coreshell fiber networks has not been fully explored. The physical and chemical parameters (e.g., electrical voltage, flow rate, distance between tip and collector, surface tension, viscoelasticity, solubility, volatility, and conductivity of solution) of electrospinning systems play an important role in controlling the morphologies of polymeric nanofibers.20,21 Furthermore, ambient parameters (e.g., humidity, temperature, and pressure) regulate phase separation and solvent evaporation processes that can control the porosity of fiber networks.22 The coreshell or hollow nanofibers containing the ceramic core and polymer shell have been generated by the electrospinning system. The selective removal of the core could generate the hollow or porous nanofibers. Although the sizes of nanofibers were homogeneous, the presence of a solgel precursor in the sheath liquid was required for creating the hollow fibers.23 Porous electrospun nanofibers have also been recently fabricated using the phase separation process.24,25 Organic solvents, such as chloroform, acetone, and dichloromethane, have been used for the phase separation process, which can create nano- or micropores. To generate porous membranes, various phase separation techniques have been used, such as thermally induced phase r 2011 American Chemical Society

separation (TIPS), air-casting of the polymer solution, immersion precipitation, and precipitation from the vapor phase.26 Recently, a phase separation-based electrospinning system has been developed to create porous nanofibers.27 This phase separation process has several advantages (e.g., low protein denaturation and low heat generation) as compared with the conventional TIPS technique. To synthesize porous nanofibers, wet electrospinning has been developed.2830 It has been used for the synthesis of three-dimensional sponge-formed nanofibers. The nanofibers synthesized by the wet electrospinning system exhibit lower bulk density and higher porosity. The water or other solvents (e.g., ethyl alcohol, tertiary-butyl alcohol, acetone, and sodium hydroxide solutions) were used as a collector. Although the porous nanofibers have been generated using the electrospinning system, the synthesis of porous core shell fiber networks has not been previously explored. Here, we fabricated porous coreshell fiber networks using the phase separation and solvent evaporation process. We hypothesized that the use of a phase separation and solvent evaporation process in a water-immersed collector would regulate the formation of porous coreshell fiber network structures. Gelatin and polycaprolactone (PCL) were used as a core and shell material, respectively. When the cells were cultured at 37 °C, the morphology of gelatin-based nanofibers changed due to its solubility in water at 37 °C. The thermal coagulation and degradation of gelatin at high temperatures may reduce the Received: April 6, 2011 Revised: July 5, 2011 Published: July 07, 2011 10993

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functionality of natural biomaterials. To overcome this limitation, the gelatin core nanofiber was encapsulated by a PCL shell nanofiber due to the higher hydrophobicity and insolubility of PCL. Therefore, this porous coreshell fiber network could be a potentially powerful tool for drug delivery, filtration, and tissue engineering applications.

2. MATERIALS AND METHODS 2.1. Materials. Gelatin (type A, MW = 50 000), polycaprolactone (MW = 80 000), and ss-mercaptoethanol were purchased from Sigma-Aldrich. Acetic acid (CH3COOH, 99.5% purity) and chloroform (CHCl3, 99.5% purity) were purchased from Smachun Chemicals, Korea. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, embryonic stem cell qualified FBS, L-glutamine, Alexa Fluor 488 phalloidin, and 40 ,6-diamidino-2-phenylindole (DAPI) were obtained from Invitrogen, U.S.A. Leukemia inhibitory factor (LIF) was purchased from Millipore. Trypsin was purchased from WelGene Company, Korea, and paraformaldehyde was obtained from Biosesang, Korea. 2.2. PCL and Gelatin Nanofiber. PCL nanofibers were fabricated by dissolving 10% PCL in acetic acid. The PCL polymer solution dissolved in acetic acid was magnetically stirred at room temperature for 4 h. An electrospinning system (NanoNC Co., Korea) was used to generate PCL nanofibers. The electrospinning system consisted of a syringe pump, a highvoltage power supply, and a grounded collector. The PCL polymer solution was loaded into a 10 mL syringe and was subsequently infused by the syringe pump at a flow rate of 0.4 mL/h into a stainless steel nozzle with a diameter of 30 GA, which was connected to the high-voltage power supply (7 kV). The distance between the nozzle and the ground collector was 12 cm. For the gelatin nanofibers, 10% gelatin was dissolved in 90% acetic acid and was magnetically stirred for 2 h at room temperature. 2.3. GelatinPCL CoreShell Nanofiber. To synthesize gelatinPCL coreshell nanofibers, 10% gelatin and various PCL concentrations (4%, 6%, 8%, 10% and 12%) were dissolved in acetic acid. Dual concentric needles (coaxial needle) and two syringe pumps were used for the coreshell nanofiber synthesis (Figure 1A). The gelatin solution was loaded into the internal capillary as a core, whereas the PCL solution was loaded into the external capillary as a shell. The applied potential difference between the needle and the collector electrode was 1016 kV for 412% PCL concentrations. The distance between the needle and collector was 12 cm. We used a constant flow rate of 0.04 mL/h for the core (gelatin) and 0.04 mL/h of the shell (PCL) to decrease the sizes of coreshell nanofibers. 2.4. Porous PCL Fiber Network. To generate porous PCL fiber network, 6% PCL was dissolved in chloroform. The ground collector was immersed in cold water (10 °C). The applied potential difference between the stainless steel nozzle and ground collector was 17 kV. We used a constant flow rate of 0.4 mL/h and a 12 cm distance between the nozzle and ground collector. 2.5. Porous GelatinPCL CoreShell Fiber Network. The porous gelatinPCL coreshell fiber networks were synthesized using a 10% gelatin solution dissolved in 90% acetic acid (core) and 6% PCL solution in chloroform (shell). The applied potential difference was 15 kV with a 12 cm distance between the nozzle and the collector electrode, which was immersed in cold

Figure 1. Electrospinning system for creating coreshell and porous coreshell fiber networks. (A) Coreshell electrospinning system for coreshell nanofibers. Scale bar is 2 μm. (B) Electrospinning setup in a water-immersed collector for porous coreshell fiber networks. Scale bar is 10 μm.

water (10 °C) (Figure 1B). We also used a constant flow rate (0.4 mL/h) to synthesize porous gelatinPCL coreshell fiber networks. 2.6. Morphological Characterization. The morphologies of the nanofibers were characterized by scanning electron microscope (SEM). The nanofiber samples were coated with platinum (5 wt % on activated carbon) using a turbo sputter coater (EMITECH, K575X). SEM images were acquired at a high voltage of 20 kV. The elements of gelatin, PCL, and gelatinPCL coreshell nanofibers were analyzed using energy dispersive X-ray spectroscopy (EDS) in field emission (FE)-SEM. The coreshell structure of the nanofibers was also confirmed by transmission electron microscope (TEM, JEM-2100F) at a high voltage of 200 kV, a dark current of 95 μA, and an emission current of 126 μA. 2.7. Cell Culture. Human breast carcinoma cell lines (MCF7) and the NIH3T3 fibroblast cells were cultured in high-glucose DMEM supplemented with 10% FBS and 1% penicillin/ streptomycin antibiotics. The mouse embryonic stem cells (R1 line) were cultured in DMEM supplemented with 10% (v/v) embryonic stem cell qualified FBS, 100 units/mL penicillin and 100 μg/mL streptomycin, 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, and 1,000 units/mL LIF. Trypsin was used to detach the cells that were adhered to a tissue culture flask. Cells were resuspended after centrifuging at 1,000 rpm for 5 min and 2  105 cells/well were seeded on 6-well plates and were subsequently cultured for 3 days. The porous fiber networks were sterilized by ultraviolet light for 30 min before culturing the cells. 2.8. Immunocytochemistry. Cells were fixed with 4% paraformaldehyde for 10 min, washed three times with phosphate buffered saline (PBS), followed by permeabilization with 0.1% Triton X-100 in PBS, and blocking of nonspecific binding with 1% bovine serum albumin (BSA) in PBS. Cells were stained with Alexa Fluor 488 phalloidin and DAPI to confirm the cytoskeleton and cell nucleus, respectively. 10994

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Figure 2. Scanning electron micrographs of gelatin and PCL nanofibers and their size profile distribution and elemental characterization. (A) Gelatin nanofibers fabricated with 10% gelatin concentration in acetic acid. Scale bar is 2 μm. (B) Size profile distribution of gelatin nanofibers fabricated with 10% gelatin concentration in acetic acid. (C) Elemental characterization of nanofibers fabricated with 10% gelatin using EDS in FE-SEM. (D) PCL nanofibers fabricated with 10% PCL concentration in acetic acid. Scale bar is 2 μm. (E) Size profile distribution of PCL nanofibers fabricated with 10% PCL concentration in acetic acid. (F) Elemental characterization of nanofibers fabricated with 10% PCL using EDS in FE-SEM.

2.9. Contact Angle Measurement. The surface wettability of the nanofibers was analyzed measuring the contact angles (Phoenix3000 Plus, Suwon, Korea). 2.10. Statistical Analysis. All data are presented as means ( standard deviations and were statistically compared using Student’s t-test. A p-value less than 0.05 or 0.01 was considered statistically significant. All error bars illustrate standard deviations.

3. RESULTS AND DISCUSSION 3.1. Gelatin and PCL Nanofibers. Gelatin and PCL nanofibers were synthesized using the electrospinning system. The SEM image and size profile analysis of the gelatin nanofibers indicated that 10% gelatin nanofibers had an average diameter of 120 ( 22.44 nm (Figure 2A,B). The gelatin solutions of higher concentrations (g12% (w/v)) could not be electrospun, because they might turn into a gel at room temperature. However, the beads-on-string structures have been observed in electrospun gelatin nanofibers at lower concentrations (68% gelatin), as previously described.31 When higher electric force was applied, the droplets formed in the electrospinning process generated beads on the nanofibers due to the lower viscosity of the gelatin. However, when the polymer concentration was increased, bead formation was reduced until smooth and bead-free nanofibers were formed, as previously described.32 Thus, uniform-sized nanofibers were synthesized at a 10% gelatin concentration. It has been established that the surface tension and viscosity of the polymer play an important role in controlling the beads-on-string or smooth morphologies of nanofiber structures.31 The viscosity of the polymer solution increased with increasing polymer concentrations. The surface tension caused the formation of beads, whereas viscoelastic forces inhibited the formation of beads, resulting in the formation of smooth nanofibers.

Therefore, we observed beads at lower polymer concentrations (low viscosities), when surface tension was higher than viscoelastic force. However, the bead formation was eliminated at higher polymer concentrations. The temperature also plays an important role in controlling the size of electrospun nanofibers. When polymer solution is electrospun at a higher temperature, the fibers with uniform diameters can be produced. This may be due to the lower viscosity of the solution and higher solubility of the polymer in the solvent. In the case of nonporous fibers, the fiber diameter can be decreased with increasing the temperature, due to lower viscosity of polymers at higher temperatures. As the functionality of the gelatin is affected by the temperature, the molecular structure may also be changed at higher temperatures. We found that higher temperatures were not suitable for the electrospinning of gelatin-based materials. Therefore, we maintained the electrospinning temperature to 25 °C for all conditions. To produce smooth, bead-free PCL nanofibers with uniform sizes, the experimental conditions were optimized by dissolving different concentrations (412%) of PCL in acetic acid. We observed that lower (12%) PCL concentrations did not lead to the formation of smooth PCL nanofibers. The SEM image and size profile analysis of PCL nanofibers synthesized at a 10% PCL concentration showed that the average diameter of the PCL nanofibers was 153 ( 26.19 nm (Figure 2D,E). Therefore, we determined that 10% PCL was the optimum concentration that could generate smooth nanofiber structures. We also analyzed the elemental morphology of gelatin and PCL nanofibers using EDS in FE-SEM. We observed that the major element of 10% gelatin nanofiber was carbon, oxygen, and nitrogen (Figure 2C), whereas the major element of 10% PCL nanofibers was carbon and oxygen (Figure 2F). 3.2. GelatinPCL CoreShell Nanofibers. To synthesize coreshell nanofibers, we used gelatin as a core and PCL as a shell material. We used 10% gelatin and various concentrations of 10995

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Figure 3. Scanning electron micrographs of coreshell nanofibers synthesized with various PCL concentrations. (A) GelatinPCL coreshell nanofibers synthesized with 10% gelatin and 4% PCL concentrations in acetic acid. Scale bar is 5 μm. (B) GelatinPCL coreshell nanofibers fabricated with 10% gelatin and 6% PCL concentrations in acetic acid. Scale bar is 2 μm. (C) GelatinPCL coreshell nanofibers fabricated with 10% gelatin and 8% PCL concentrations in acetic acid. Scale bar is 2 μm. (D) GelatinPCL coreshell nanofibers fabricated with 10% gelatin and 10% PCL concentrations in acetic acid. Scale bar is 2 μm. (E) GelatinPCL coreshell nanofibers fabricated with 10% gelatin and 12% PCL concentrations in acetic acid. Scale bar is 2 μm. (F) Elemental characterization of coreshell nanofibers fabricated with 10% gelatin and 10% PCL concentrations using EDS in FE-SEM.

PCL (4%, 6%, 8%, 10%, and 12%). SEM images of gelatinPCL coreshell nanofibers showed that the unstable nanofiber morphology with several beads-on-string structures was observed at the surface of coreshell nanofibers at 4%, 6%, and 8% PCL concentrations (Figure 3AC). In contrast, uniform and beadfree smooth coreshell nanofibers were obtained at 10% PCL concentrations (Figure 3D). Size profile analysis of the core shell gelatinPCL nanofibers indicated that the coreshell nanofibers (10% gelatin:4% PCL) showed an average diameter of 105 ( 30.80 nm (Figure 4). Although the lower PCL concentration (4%) generated coreshell nanofibers with smaller sizes, a few beads were observed on the nanofibers due to the unstable cone jet formation, as previously described.31 We observed that the sizes of gelatinPCL coreshell nanofibers were directly proportional to PCL concentrations, showing that the average diameter of coreshell nanofibers with 6% and 8% PCL concentrations was 117 ( 16.56 and 132 ( 12.63 nm, respectively (Figure 4). At 10% gelatin and 10% PCL concentrations, the coreshell nanofibers showed the average diameter of 141 ( 9.23 nm and the nanofiber morphology was smooth without any beads on the coreshell nanofibers. In contrast, the higher concentration of PCL (12%) showed larger average diameter (210 ( 48.65 nm), and their sizes were nonuniform (Figure 3E). It might be due to the clogging of the dual concentric nozzles resulted from the higher viscosity at a 12% PCL concentration. We also characterized the elemental morphology of coreshell nanofibers synthesized by 10% gelatin and 10% PCL concentrations. For the coreshell nanostructure with 10% gelatin and 10% PCL concentrations, we found carbon, oxygen, and nitrogen, because coreshell nanostructures were

Figure 4. Size profile distribution of gelatinPCL coreshell nanofibers.

synthesized using gelatin and PCL (Figure 3F). The coreshell structure of the gelatinPCL nanofibers was further confirmed by TEM (Figure 5), indicating that the hydrophilic gelatin core nanofibers were encapsulated within the hydrophobic PCL shell nanofibers. We also analyzed the elemental morphology of the core and shell structures separately using EDS in FE-SEM, showing that we observed carbon, oxygen, and nitrogen in the gelatin core, whereas carbon and oxygen were found in the PCL shell. 10996

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Langmuir 3.3. Porous GelatinPCL CoreShell Fiber Networks. Porous fiber networks are of great interest in tissue engineering, drug delivery, and controlled release applications. We synthesized porous PCL fiber networks using 6% PCL in chloroform (Figure 6B). The porous PCL fiber network was fabricated using a water-immersed collector. The average diameter of the pores produced on the PCL fiber networks was 1.57 μm (Figure 6D). We also measured the aspect ratio (width/length) of the pores created on the PCL fiber network. The quantitative analysis of aspect ratios showed an average aspect ratio of 2.86, indicating highly elongated pores on the PCL fiber network (Figure 6E).

Figure 5. (A) Transmission electron micrographs to verify the formation of coreshell gelatinPCL nanofibers. (B) Elemental characterization of the core and shell fiber structure.

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After optimizing the conditions used to formulate porous PCL fiber networks, we synthesized porous coreshell fiber networks using 10% gelatin in acetic acid as a core and 6% PCL in chloroform as a shell. To generate pores on the coreshell fiber networks, we used a water-immersed collector. The mean diameter of the pores generated on the coreshell gelatinPCL fiber networks was approximately 1.04 μm (Figure 6D). The pore morphology of the coreshell fiber networks was circular (Figure 6C), in contrast with the elongated pores of the PCL fiber networks (Figure 6B). The aspect ratio of the pores generated on the coreshell fiber networks was 1.07, indicating that the pores were circular in shape (Figure 6E). The circular morphology of the pores generated on the coreshell fiber networks might be due to the diffusion of acetic acid into the PCL shell and the solvent distribution on the shell surfaces of the coreshell fiber networks. To generate porous fiber networks, we used a highly volatile solvent, such as chloroform, instead of acetic acid. When the water-immersed collector was not used, we did not find any pores on the gelatinPCL coreshell fiber networks (Figure 6A). During the solvent evaporation process, the thermodynamic instability of the polymer solution caused phase separation, as previously described.24 We demonstrated that the pores on the polymeric fiber networks were generated by the phase separation and solvent evaporation process. After the phase separation process, the polymer-rich phase was solidified, whereas pores were created by a solvent-rich phase. Another mechanism for generating pores on the nanofibers was rapid solvent evaporation, as previously described.33 The water droplets were encapsulated by polymer particles in a water-immersed collector due to

Figure 6. Porous fiber network and pore size profiles. (A) SEM image of coreshell nanofibers generated with 10% gelatin:6% PCL in chloroform without a water-immersed collector. Scale bar is 500 nm. (B) SEM image of porous fiber network generated with 6% PCL in chloroform using a waterimmersed collector. Scale bar is 10 μm. (C) SEM image of porous coreshell fiber network synthesized with 10% gelatin:6% PCL in chloroform using a water-immersed collector. Scale bar is 10 μm. (D) Pore size comparison of porous PCL fiber network and porous gelatinPCL coreshell fiber network. (E) Aspect ratios (width/length) of porous PCL fiber network and porous gelatinPCL coreshell fiber network (*p < 0.05). 10997

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Langmuir evaporative cooling. As a result, we demonstrated that the phase separation and solvent evaporation process generated in the water-immersed collector played an important role in manipulating the pore formation on coreshell fiber networks. Furthermore, the relative humidity enabled the control of the porosity of the nanofibers. It has been reported that liquidliquid phase separation could manipulate the rate of polymer solidification.26 This phase separation process with higher humidity conditions gave rise to generate numerous pores on the coreshell fiber networks. Therefore, the phase separation and solvent evaporation process enabled the control of the pores on the coreshell fiber networks. 3.4. Cell Culture on Porous CoreShell Fiber Networks. The surface wettability of nonporous nanofibers and porous fiber networks was analyzed measuring the contact angles (Figure 7). The nanofibers created with 10% gelatin concentrations showed the hydrophilic surface (44.1°), whereas the surface of nanofibers synthesized with 10% PCL concentrations was hydrophobic

Figure 7. Surface wettability of nanofibers and porous fiber networks (**p < 0.01).

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(118.3°). We observed that the coreshell nanofibers containing hydrophilic gelatin core and hydrophobic PCL shell showed the hydrophobic surface. The hydrophobic PCL surface of core shell nanofibers could prevent the dissolution and degradation of the gelatin core, as previously described.34 To examine the biological feasibility of the porous gelatinPCL coreshell fiber networks, we cultured various cells (e.g., embryonic stem cells, breast cancer cells, and fibroblast cells) on porous gelatinPCL coreshell fiber networks (10% gelatin:6% PCL). The cells cultured for 3 days on porous gelatinPCL coreshell fiber networks were highly viable (Figure 8A). When the cells were not adhered on porous gelatinPCL coreshell fiber networks, they could be dead. Furthermore, we observed the morphology of cells cultured on the porous gelatinPCL coreshell fiber networks (Figure 8B). SEM images revealed that the cells were adhered on porous gelatinPCL coreshell fiber networks. The cells can be attached to the nanofiber surfaces by the covalent bonding. The carboxyl group of PCL plays an important role in controlling the covalent bonding to increase the cell attachment on the nanofiber surfaces, as previously described.35 Furthermore, the cell attachment may be improved by the physical entrapment or focal adhesion.35

4. CONCLUSIONS We fabricated porous gelatinPCL coreshell fiber networks using an electrospinning system containing a water-immersed collector. The sizes of the gelatinPCL coreshell nanofibers were directly proportional to PCL concentrations. We demonstrated that the phase separation and solvent evaporation process generated in a water-immersed collector enabled the control of pore shapes on the fiber networks. The pores on fiber networks synthesized from PCL alone were elongated, whereas the pores created on the gelatinPCL coreshell fiber networks were circular in shape. We also measured surface wettability of nanofibers, indicating that the surface of coreshell nanofibers showed the hydrophobic property. Furthermore, immunocytochemistry showed that cells cultured for 3 days on porous

Figure 8. Fluorescent and SEM images of the cells cultured on porous gelatinPCL coreshell fiber networks (10% gelatin:6% PCL); (A) Fluorescent images of the cells cultured on porous gelatinPCL coreshell fiber networks. Scale bars are 300 μm. (B) SEM images of the cells cultured on porous gelatinPCL coreshell fiber networks. Scale bars are 50 μm (left image) and 100 μm (middle, right images). 10998

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Langmuir coreshell fiber networks were highly viable. Therefore, this porous coreshell fiber network could be a potentially powerful tool for tissue engineering applications.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

’ ACKNOWLEDGMENT This paper was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (Grant Nos. 20100004869 and R11-2008-044-01002-0) and Korea Industrial Technology Foundation (KOTEF) through the Human Resource Training Project for Strategic Technology. This research was also supported by grants from Korea Research Institute of Bioscience and Biotechnology (KRIBB) Open Innovation Program. Muhammad Gulfam was also sponsored by ‘Higher Education Commission (HEC), Govt. Of Pakistan” under the scholarship program titled: MS Level Training in Korean Universities/Industry, bearing GRE type Test Roll Number. ’ REFERENCES

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