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The core-shell nanofiber with special secondary structures like pores on the mat could be of enormous importance in biomedical as well as in biochemical fields. In this article, we have successful electrospun the core-shell nanofiber with pores on the surface. First we optimized the processing conditions for the fabrication of normal nanofiber using gelatin and PCL. Afterward, we synthesized core-shell nanofiber using gelatin as core while PCL as shell nanostructure. Subsequently, we effectively produced the core-shell fiber with special pores on the surface using phase separation process. The diameter of the simple gelatin nanofiber was observed to be less than PCL as well as gelatin-PCL core-shell fiber. The pores created on PCL as well as on gelatin-PCL core-shell fiber mat were in micrometer size. Pores on PCL fiber were observed to be elongated (foot shape) while on that of gelatin-PCL fiber was mostly circular in shape as revealed by scanning electron microscopy. In this way we have further increased the surface area of core-shell fiber mat which is the result of highly volatile solvent and phase separation process using water immersed collector. These porous bicomponent core-shell nanostructures could be used in various biomedical applications in future.
Electrospinning has attracted marvelous interests in the research society from last few decades. As being simple, versatile and straightforward technique, nano to micrometer size fibers can be synthesize precisely by electrospinning.1 The pilot scale processing of such nanostructures can be achieved with well versatility and leniency of process using this approach.2 Electrospun fibers have smaller pores and greater surface area than ordinary fibers, and have been efficiently applied in diverse fields, such as, nanocatalysis, tissue engineering,3 scaffolds, protective clothing, filtration, biomedical, pharmaceutical, optical electronics, healthcare, biotechnology, defense, security, and environmental engineering4,5.
In particularly, this is a relatively robust and simple technique to produce nanofibers from a wide variety of polymers. Spun nanofibers present a number of advantages, for instance, tremendously high surface-to-volume ratio, tunable porosity, malleability to conform to a wide variety of sizes and shapes and the ability to control the nanofiber composition to achieve the desired results from its properties and functionality. Owing to these advantages, electrospun nanofibers have been extensively investigated in the past several years for its use in various applications, such as filtration, optical and chemical sensors, electrode materials and biological scaffold, 6 7, drug delivery,8 9,10, conductive nanowires, 2, nanosensors, 11, biochemical protective clothing for the military, 12 and wound dressing, 13
The process of electrospinning involves an electric field to convert polymer solution or melt into a fiber form. Briefly, when an external electrostatic field is established between a pendant droplet of polymer solution and a metal collection device serving as a counterelectrode, the coupling of the surface charge and the external electric field creates a tangential stress. This stress results in the deformation of the droplet into a conical shape (Taylor cone). When the electric field strength exceeds a critical value needed to overcome the surface tension, the apex of the cone ejects a fluid jet toward the metal collection device. This fluid jet undergoes significant bending instability and elongation. Meanwhile, the solvent involved evaporates quickly and submicron-size fibers or nanofibers in nonwoven form are deposited on the collection device.1,9,10,14-16
Although, fabrication of nanofibers and nanostructures encompassing single or blended materials have been broadly studied, however, synthesis of coaxial compound and core-shell nanostructures are getting more importance due to their novel and unique properties.17 Such core shell nanostructures find their applications in the protection of biologically or chemically unstable active substances from adverse environmental conditions, sustained delivery of biomolecular drugs, surface modification while unaffecting the natural functionality of core nanostructure, and to prevent the decay of a vulnerable compound under certain circumstances.18 The core shell nanostructured fibers can be fabricated in a variety of ways such as self-assembly,19 laser ablation,20 template synthesis,21 and with tubes by fiber templates (TUFT) method based on electrospinning.22
Although, the fabrication of core-shell nanofibers from coaxial electrospinning have been already described23, however, the processing details and the potential applications of those nanofibers have not yet been fully explored. Special secondary features have also been created on nanofiber recently and hollow to porous nanofibers have successfully been electrospinned to attain unique properties of these nanostructures.24 Introduction of porous micro to nanostructures on individual fiber or on nonwoven mats of nanofibers could result into the further amplification in surface area of these tiny creatures. Numerous applications can be obtained by increasing the surface area which includes filtration, catalysis, absorption, solar cells, fuel cells, batteries, tissue engineering, and drug delivery.25,26 In particular, enhanced drug loading can be possible, because porous surfaces can offer more incorporation or adsorption sites for drug loading. Additionally, surface modification of nanofiber can be performed by creating porous structures which can ultimately result into enhanced cellular adhesion providing more anchoring points for cells accommodation and mutual interactions with scaffold materials to mimic the natural extracellular matrix and it also facilitate the diffusion of nutrients,27 and hence, can be used in tissue engineering.28
Several electrospinning parameters are responsible for the inimitable morphology of polymer fibers, which include the applied electrical voltage, solution flow rate, distance between tip and collector, and polymer solution properties such as surface tension, viscoelasticity, polymer solubility, volatility, and conductivity of solution, The ambient parameters such as humidity, temperature, and pressure also play a vital role to control the morphology of electrospun nanofiber. The competition between the rate of phase separation and solvent evaporation is also an imperative factor.29,30 Currently, most of the porous electrospun nanofibers are fabricated typically with phase separation technique. 28,31,32 Complex and manifold organic solvent systems are prerequisite of such a phase separation process which results into nano or micropores. It is also critical to note that a single solvent cannot dissolve all materials of interest for electrospinning. So, it is quite complicated to accurately regulate the rates of phase separation and solvent evaporation to facilitate the formation of pores on nanofibers. The abovementioned limitations of such a scheme give rise to a restricted choice of polymers and drugs in potential applications. 28
Currently, thermally induced phase separation (TIPS) technique has been developed to generate porous polystyrene (PS) membranes33, however, this technique cannot be used in polypeptide based biomaterials because heat processing can impair the functionality of these biopolymers to a great extent. More recently, phase separation electrospinning system collaborated with water immersed collector has also been used to create porous nanofiber mats 34. This technique provides efficient and large scale production of such porous scaffolds.
Although, simple porous nanofibers from various polymers have been produced as reported earlier, but core-shell-porous fibers have not yet been produced according to our knowledge. By combining the both nanostructures (i.e. pores and core-shell) on a single fiber or on nonwoven fiber mats, can result into a unique morphology which can be used in various applications like drug delivery, tissue engineering, filtration, sensing and biocatalysis fields. We hypothesize that the bicomponent core-shell structure with pores on surface can enhance the surface area of fiber to a large extent while the encapsulated active component (core e.g. drug or protein) can be delivered to the preferred site of application with more precision and accuracy.
In this article, we have generated pores on the core-shell nanofiber mat using gelatin as a core and polycaprolactone (PCL) as a shell materials. Gelatin is a natural biopolymer which is obtained from controlled acidic or alkaline pretreatment of collagens.1 Owing to its biological derivation, excellent biodegradability and biocompatibility, gelatin has been profusely used in pharmaceutical and biomedical fields as sealants for vascular prostheses,35, carriers for drug delivery,36 and dressings for wound healing.37 Instead of its aforementioned merits, gelatin is hardly conceded as an active material for tissue engineering purpose without any special treatment, due to its solubility as colloidal solution in water at or above 37°C and coagulation into gel at lower temperature.38 As most of the cell culture techniques are carried out at 37 °C, the gelatin nanofiber can lost its morphology because of being soluble in water at this critical temperature. Although, thermal crosslinking can be performed to overcome this problem, but thermal coagulation and degradation at higher temperature can lead to biodeactivation of this compound. To overcome this problem we proposed encapsulation of gelatin fiber into PCL shell because of its higher hydrophobicity and insolubility in water.39 Furthermore, we have created pores into this core-shell gelatin-PCL nanofiber for enhanced biochemical and biological applications. We hypothesize that this novel idea of pore generation on core-shell nanofiber can further enhance the surface area while not affecting the core-nanostructures. These core-shell porous nanostructures can have potential applications in drug delivery, filtration, catalysis and tissue engineering fields which can result into further up gradation of these vital areas of science and engineering.
2. Materials and methods
Gelatin (type A), and polycaprolactone (PCL), (Mw=80000) were purchased from Sigma-Aldrich. Acetic acid (CH3COOH, purity= 99.5 %), and chloroform (CHCl3, purity= 99.5 %) were procured from Smachun chemicals, Korea. The electrospinning system supported with normal as well as core-shell needles was obtained from NanoNC Company, Korea.
2.2. Fabrication of PCL and gelatin nanofibers
Polycaprolactone nanofiber was fabricated by dissolving 10% PCL in acetic acid Before the production of PCL nanofiber by electrospinning, the PCL polymer solution dissolved in acetic acid was magnetically stirred at room temperature for 4 h.
The normal electrospinning system for the fabrication of PCL normal nanofiber is sown in fig. 1(A).The electrospinning system consisted of a syringe pump loaded with a syringe, a high-voltage power supply and a grounded collector. The PCL polymer solution was loaded in a 10 mL syringe and was continuously pushed by the syringe pump at a flow rate of o.4 mL/h to a stainless steel nozzle with a diameter of 30 GA, which was connected to the high-voltage power supply to generate a 7 KV potential difference between the nozzle and the grounded stainless steel foil/sheet. The distance between the nozzle and the ground collector was set to 12 cm after optimization. For the fabrication of gelatin nanofiber, 10% gelatin was dissolved in 90% acetic acid and magnetically stirred for 2 h at room temperature. The other processing conditions were same as in case of PCL nanofiber except the electrical potential difference which was sustained at 14 KV.
2.3. Fabrication of gelatin-PCL core-shell nanofiber
For the fabrication of gelatin-PCL composite core-shell nanofiber, 10% gelatin was dissolved in 90% acetic as described above and different concentrations of PCL such as 4%, 6%, 8%, 10% and, 12% were prepared by dissolving PCL in acetic acid and by magnetically stirrering at room temperature for 4 h. The electrospinning system for the fabrication of gelatin-core shell composite nanofiber is shown in figure 1(B). For core-shell composite nanofiber fabrication, the dual concentric needle (coaxial needle) was used and a supplementary syringe pump fitted with a syringe was used. The coaxial needle comprise of two capillaries, the external capillary and an internal capillary. The outer diameter (OD) of external capillary (22G) was 0.70 mm and internal diameter (ID) was 0.40 mm). The internal capillary (30 G) consisted of 0.30 mm outer diameter and 0.15 mm of internal diameter. The gelatin solution was loaded into a 10 mL syringe attached to internal capillary as core while PCL solutions was loaded into 10 mL syringe attached to external capillary as shell. The applied potential difference between needle and collector electrode was 17, 16, 15, 13 and 12 KV for 4%, 6%, 8%, 10%, and 12% PCL concentrations respectively. In all core-shell nanofiber production experiments, the needle to collector distance of 12 cm was maintained, while a constant flow rate of 0.4 mL/h of core (gelatin) and 0.4 mL/h of shell (PCL) was manipulated
2.4. Fabrication of PCL porous fiber
For the fabrication of porous PCL fiber, 6% PCL was dissolved in chloroform (CHCl3, Smachun chemicals, Korea, purity= 99.5 %). The electrospinning system was normal electrospinning system as described previously except the ground collector which was immersed in cold water (10 0C). The applied potential difference between the stainless steel nozzle (32G) and ground collector was 17 KV with a constant flow rate of 0.4 mL/h and a distance of 12 cm between nozzle and ground collector.
2.5. Fabrication of gelatin-PCL core-shell porous fiber
The gelatin-PCL core-shell porous fiber was fabricated by using 10% gelatin solution dissolved in 90% acetic acid (core) while 6% PCL solution in chloroform (shell). The applied potential difference was 15 KV with a distance of 12 cm between nozzle and collector electrode which was immersed in cold water (10 0C). The flow rate of gelatin and PCL was maintained to constant 0.4 mL/h respectively. The dual concentric needle was the same as used for the gelatin-PCL core-shell fiber fabrication.
2.6. Fiber morphology characterization
The morphology of nanofiber was characterized by a scanning electron microscope (TESCAN Model: SEM VEGA/SBH Motororize).The nanofiber samples were coated with platinum coating (5 wt% on activated carbon) with the help of a turbo sputter coater (EMITECH: K575X/Carb Peltier Cooled) before scanning electron microscopy. The SEM images were taken at a high voltage of 20 KV.
2.7. Core-shell verification
The core-shell structure of nanofiber was confirmed with the help of a transmission electron microscope (TEM), (JEM-2100F) at a high voltage of 200 KV and a dark current of 95 µA and 126 µA of emission current.
2.8. Cell culture
The human breast carcinoma cell lines MCF7 and the mouse embryonic fibroblast cell lines NIH3T3 were grown in high glucose Dulbecco's Modified Eagle's Medium (DMEM, Invitrogen, USA), supplemented with 10% fetal bovine serum and 1% pen/strep antibiotic. The mouse embryonic stem cells R1 were cultured in high glucose-Dulbecco's Modified Eagles Medium (DMEM; Gibco) supplemented with 10% (vol/vol) ES qualified-FBS (Gibco), 100 U/mL penicillin and 100 ãŽ/mL streptomycin (Gibco), 1 mM L-glutamine (Gibco), 0.1 mM ß-mercaptoethanol (Sigma-aldrich), and 1,000 U/mL of leukemia inhibitory factor (LIF; Chemicon) in an incubator (5% CO2, 37â„ƒ). Trypsin was used to detach the cells adhered to a tissue culture flask. Cells were resuspended after centrifuging at 1,000 rpm for 5 min and 2 X 105cells/well was seeded on 6well plate and cultured for 3days. Before cell culturing, the nanofiber scaffolds were sterilized by exposing the nanofiber scaffolds to UV-light for 30 min.
2.9. Morphological observation and cell viability
Cells were fixed with 4% paraformaldehyde (Biosesang, Korea) for 10 min, washed three times with PBS, followed by permeabilization with 0.1% triton X-100 in PBS, and blocking of nonspecific binding by incubation with 1% BSA (agdia, USA) in PBS. Cells were stained with Alexa Fluor 488 phalloidin (Invitrogen, USA) and were counterstained with DAPI (Invitrogen, USA) to visualize the cell nucleus.
2.10. Statistical analysis
All data were expressed as mean ± SD and were statistically compared by student t-test where necessary. The 5% significance level (p-value equal to or less than 0.05) was considered to be statistically significant unless otherwise noted. All error bars were presented as standard deviations.
3. Results and discussion
3.1. Gelatin nanofiber
Gelatin nanofiber was fabricated to analyze the individual behavior of gelatin nanofiber for cell culture and size profile characterization. We optimized the conditions for the fabrication of gelatin nanofiber by using different concentrations of gelatin in 90% acetic acid and found that 10% gelatin in 90% acetic acid is optimum for the production of gelatin nanofiber as also described previously.40 16 The scanning electron microscopic image and size profile distribution of gelatin nanofiber are illustrated in Fig. 2 (A) and Fig. 2(C) respectively. It is revealed that most of the gelatin nanofibers were in the range of 100 nm to 140 nm with an average diameter of 120 ± 22.44 nm. These optimized conditions of nanofiber fabrication resulted into the production of bead free nanofibers. The smooth and steady fibrous structure can be obtained only beyond a critical concentration and under process limiting viscosity in the electrospinning of polymers with a certain molecular weight. At room temperature, the gelatin solutions of 12% (w/v) concentration and above can be turned into gel, so they could not be electrospun, while the beads-on-string structures have been observed in electrospun fibres of gelatin at lower concentration of 6% and 8%..40 When applying high electric force, the development of droplets in electrospray results into the formation of beads as in case of low-molecular-weight liquid. However, in the case of a polymer solution, a filament of beads linked by a fibre is formed because the emerging jet remain stabilize and does not break up into droplets. With further boost up polymer concentration, bead formation is diminished until smooth and steady fibres are formed.41 In this study, uniform fibres were synthesized by increasing the gelatin concentration to 10%.The struggle between surface tension and viscosity of the polymer play a vital role in this change from beads-on-string structures to steady and smooth fibres.40 By increasing the polymer concentration, the viscosity of the polymer solution increased. The surface tension caused the formation of beads, while the viscoelastic forces resisted the formation of beads and allowed for the formation of smooth fibres. Therefore formation of beads at lower polymer solution concentration (low viscosity) occurred when surface tension had a greater effect than the viscoelastic force. However, bead formation was reduced and finally eliminated at higher polymer solution concentration, where viscoelastic forces overtook surface tension.
3.2. PCL nanofiber
To produce bead free PCL nanofiber with a uniform distribution of diameter, the experimental conditions were optimized by dissolving different concentration of PCL in acetic acid. We found that PCL concentration of less than 4% and higher than 12% are not optimum for the production of PCL nanofiber. The 10% concentration of PCL in acetic acid is found best for PCL nanofiber fabrication. The scanning electron microscopic image and size profile distribution of PCL nanofiber at 10% PCL concentration is shown in Fig.2 (C) and Fig.2 (D) respectively. Most of the size was distributed in the range of 140-160 nm with a mean diameter of 153±26.19 nm.
3.3. Gelatin-PCL core-shell nanofiber
For the production of core-shell nanofiber we used gelatin as core while PCL as a shell material. The gelatin concentration was kept constant to 10% as optimized in normal gelatin nanofiber production experiment while the concentration of PCL was varied to 4%, 6%, 8%, 10%, and 12%. The scanning electron microscopic images of gelatin-PCL core-shell nanofiber at 4%, 6%, and 8% concentration, and size profile distributions are shown in Fig.3 (A-F). In case of 10% gelatin and 4% PCL concentration, the most of the size profile of core-shell nanofiber was in the range of 140 nm to 160 nm with an average diameter of 138± 20.36 nm. This lower concentration of PCL resulted into core-shell nanofiber of small size but there were some beads in the nanofiber matrix due to unstable cone jet formation as also reported earlier.40 The 6% PCL concentration resulted into core-shell nanofiber which was mostly dispersed in the range of 120-180 nm with an average diameter of 155±25.36 nm. We revealed that 8% concentration of PCL generated the nanofiber having a size profile distribution of mostly 160 nm to 220 nm with an average diameter of 165±38.75 nm. From this analysis we revealed that the size of gelatin-PCL core-shell nanofiber is directly proportional to the PCL concentration while keeping the gelatin concentration constant (10%).
Furthermore, we revealed that the 10% PCL concentration is optimum for the fabrication of gelatin-PCL core-shell nanofiber while keeping the gelatin concentration constant to 10%. At 10% gelatin and 10% PCL concentration, the core shell nanofiber has a size profile distribution of 200 nm to 320 nm. The mean diameter at this concentration was observed to be about 194 nm±39.18 nm as illustrated in fig. 4(A-B). There were no beads appeared at this optimized concentrations of polymers. The higher concentration of PCL up to 12 % produced the nanofiber with highly dispersed size profile in the range of 220-520 nm mostly. The mean diameter was 335 nm and with higher standard deviation of 130.4 nm. The morphology of the nanofiber at this higher polymer concentration was not uniform. The whole length of even a single nanofiber was not uniformly distributed. This may be due to the possible clogging of dual concentric nozzle of coaxial electrospinning system by higher concentration of PCL. We revealed that, keeping the gelatin concentration up to 10% constant, higher the PCL concentration, higher was the diameter of core-shell gelatin nanofiber. The core-shell verification of gelatin-PCL nanofiber was confirmed by transmission electron microscope (TEM) made at 10% gelatin and 8% PCL. The Fig.5 (A) demonstrates the core-shell structure of gelatin-PCL nanofiber acquired by the TEM. The core structure of gelatin appeared as shinning region due to more nitrogen contents in gelatin.
3.4. PCL porous fiber
Porous scaffolds are very important in tissue engineering and drug delivery and controlled release. We have produced the PCL porous fiber using 6% PCL in chloroform. The porous PCL nanofiber was fabricated by using water immersed collector. Fig.6 (A) shows the PCL porous fiber fabricated with 6% PCL concentration in chloroform. The average diameter of pores produced on the surface of PCL nanofiber mat was 1570 nm as shown in fig. 6 (C). The SEM images of porous fiber mat revealed that most pores were elongated in shape and resembled with the shape of foot. We also calculated the aspect ratio (width/length) of pores produced on the PCL fiber. The results of aspect ratio are demonstrated in the fig.6 (D) which show that the pores have an average aspect ratio of 2.86 which revealed the highly elongated shape of pores produced on PCL fiber mat.
3.4. Gelatin-PCL core-shell porous fiber
The production of porous core-shell bicomponent nanofiber is a novel idea for the production of unique scaffolds having various applications in tissue engineering, drug and protein delivery and controlled release. We have fabricated the porous core-shell fiber using 10% gelatin in acetic acid as core while 6% PCL in chloroform as shell. The collector electrode was immersed in water to produce pores. The SEM image of porous core-shell gelatin-PCL fiber is shown in Fig. 6(B). The mean diameter of pores produced on the surface of core-shell gelatin-PCL fiber mat was about 1040 nm. The pore morphology was mostly round shape as compare to pores produced on the surface of normal PCL fiber mat. The aspect ratio of pores was about 1.07 which revealed the round shape of pores as shown in Fig. 6(D).
For the fabrication of porous nonwoven fiber mats, highly volatile solvent is required,42 therefore, we have used chloroform instead of acetic acid. Without water immersed collector, the chloroform evaporates rapidly, and results into less polymer rich phase and high solvent rich phase leaving almost no pore on the nanofiber.43 During solvent evaporation, the thermodynamic unstability of polymer solution causes the phase isolation which results into a polymer rich and a polymer poor phase32. The pores in the polymer fiber mat are generated due to this important phase separation process. The schematic mechanism of pore generation on nanofiber mats is demonstrated in fig.7. After phase separation, the concentrated polymer phase solidified into fiber while pores are produced by polymer poor phase. Another reason for the production of pores in the nanofiber produced by electrospinning system is the rapid solvent evaporation which causes the evaporative cooling which considerably diminishes the temperature of electrospinning jet.31 As a consequence, the polymer particles encapsulated water droplets either from the water bath due to evaporative cooling during spinning or from atmospheric moisture. The drying of the nanofiber mat resulted into the generation of pores constructed by the imprints of water droplets on the nanofiber surface. This is why the pores are produced on the nanofiber surface and water and volatile solvents play an important role in the mechanism of pore formation.42 This phase separation process aided with high moisture conditions gave rise to two distinct phases a "sea" and an "island" in the form of a web,26 the sea phase is characterized by pore walls and the island is denoted by frequent pores along the length of nanofiber mats.44
3.5. Cell culture on nanofiber
This work explored the fabrication of gelatin, and PCL individual as well as core-shell and porous core-shell nanofibers to a wider horizon. After optimizing the conditions for individual and core-shell fibers, the pore generation was performed on the core-shell nanofiber mats. In case of gelatin-PCL core-shell nanofiber, increasing the PCL concentration resulted into the nanofiber with higher diameter and with greater poly dispersity while maintaining the concentration of gelatin (10%) and other processing conditions constant. The pores on the individual PCL fiber mat were observed to be elongated in morphology while in case of gelatin-PCL porous core-shell nanofiber mats, the pore morphology was appeared almost circular in nature. The pore generation on nanofiber mat is due to the phase separation process supported with higher humidity conditions which was provided by water immersed collector. This bicomponent core-shell porous scaffold could be potentially useful in tissue engineering, filtration, and drug delivery. (Conclusion part about cell culture remaining)