Optimum parameters for the production of nano-scale electrospun polycaprolactone to be used as a biomedical material

Electrospun nano-polycaprolactone (PCL) is an ideal candidate for biomedical applications, as it mimics the extracellular matrix and possesses good biocompatibility, biodegradability and mechanical properties. Formic acid/acetic acid (FA/AA) and formic acid/acetone (FA/A) solvent systems were reported to be the safest solvents for the preparation of nanoscale electrospun polycaprolactone and they are also a common solvent with many natural polymers. In the present study a comparison between the electrospun fibers produced by the two systems was done. The optimum conditions for preparing PCL nanofibers were studied. The results indicated that finer fibers were found for formic acid/acetic acid in comparison with those produced by formic acid/acetone solvent system. In addition, it was found that optimum conditions for PCL nanofibers electrospinning were detected for 70:30 FA/AA solvent ratio with 15% PCL concentration and a tip to collector distance of 12.5 cm at 20 kV.


Introduction
Electrospun membranes constitute a broad range of materials with a number of applications from biomedical to environ mental fields [1]. Membranes from nanofibrous materials mimic the extracellular matrix as they are characterized by a high surface area to volume ratio and the presence of mul tiporous structure [2]. Electrospinning has attracted tremen dous attention as it provides a simple and versatile method for fabricating nanofibers from a rich variety of polymers or polymers loaded with nonspinnable materials, so allowing a wide variety in design in of functional membranes [3,4]. A typical electrospinning setup contains a high voltage DC (or AC) supply (usually in the range of 10-50 kV), a spinneret with a metallic needle, and a collector [5]. High voltage is applied between the metallic needle and the collector. As a result, a drop of the polymer solution from the spinneret will be electrified to form a Taylor cone. By increasing the electric force, the Taylor cone will overcome the surface tension and then undergo a continuous elongation forming the electrospun fibers on the collector [5].
Recently, nanofibrous mats of polycaprolactone have been of a great interest in scientific literature focusing on scaf folds for tissue engineering [6][7][8]. Polycaprolactone (PCL), Advances in Natural Sciences: Nanoscience and Nanotechnology Optimum parameters for the production of nano-scale electrospun polycaprolactone to be used as a biomedical material a semicrystalline linear synthetic hydrophobic polymer, is one of the most commonly used polymers in electrospinning either alone or in combination with other additives to fabricate nanofibrous mats for many biomedical applications [9]. For medical applications, it is somehow essential to have a fiber diameter in the nanorange in order to mimic the extracel lular matrix (ECM) morphology to optimize cell growth [10]. PCL was often produced by chloroform but it produces rather microfibers instead of nanofibers as the diameters were in the range from 3 to 5 µm [11]. PCL nanofibers were also pro duced by other relatively highly toxic solvents and relatively costlier solvents, such as dimethylformamide (DMF), tetra fluoroethylene, methylene chloride, dichloroethane and pyri dine but some of them lacks stability and reproducibility [12]. NanoPCL fibers were produced successfully by safer solvent mixtures as formic acid/acetone (FA/A) and formic acid/acetic acid (FA/AA) systems [12,13]. Both solvent systems were reported as the safest solvent systems [12,13] and also they are a common solvent of polycaprolactone with other poly mers such as chitosan, gelatin and collagen [14][15][16][17]. During the research work, a comparison between the fibers produced by the two systems was done. In addition, the optimum con ditions for PCL electrospinning in the system with the best attained results were investigated.
where η is the viscosity after the determined time and η 0 is the initial viscosity of the solution.

Results and discussion
3.1. Solvent study 3.1.1. Fiber morphology The scanning electron microscopy images of the produced fibers from formic acid/acetic acid (FA/AA) and formic acid/acetone (FA/A) solvent systems were shown in figure 1. From the figure, it was shown that the fibers produced from FA/AA solvent system mostly occur in a range less than 100 nm which is much finer than that produced by FA/A solvent. This might be attributed the additional acidic hydrolytic effect of acetic acid. Therefore, FA/AA solvent system was selected for further studies.

Rheological measurements
The viscosity values of the two polymeric solvent systems were illustrated in table 1.
The change in the values of the viscosity with time for both solvent systems was shown in figure 2. It is found that FA/AA solvent system showed less stability than FA/A solvent system, as acetic acid was involved in the acidic hydrolysis of PCL which may fasten its hydrolytic degradation.

Conductivity measurements
The conductivity values of the two solvent systems are clarified in table 1. Both sol vent systems showed nearly equal conductivity values. It is noticed that FA/AA solvent system revealed a slightly higher conductivity value.

Fiber morphology
The scanning electron microscopy images of the produced fibers for 70:30, 50:50, 30:70 and 10:90 FA/AA solvent ratios, as well as acetic acid, were shown in figure 3. Formic acid and 90:10 FA/AA samples were not viscous enough as presented in table 2 and hence they were unstable to produce fibers. The polymeric solutions were thus electrosprayed rather than been electrospinned in both solvent systems. A high ratio of formic acid causes a major influence on the electric field applied during electro spinning causing solvent molecules reorientation. This leads to instability in electrospinning and therefore adding ace tic acid will help to overcome this instability. These results were in agreement with the study of Van der Schueren [12]. However, by increasing the acetic acid ratio to more than 50%, a decrease in the electrospinning reproducibility was observed. The mean fiber diameter showed an increasing trend by increasing acetic acid ratio as observed in table 2 and figure 3. This may be attributed to the increase of viscos ity and decrease in conductivity [18].

Rheological measurements
The viscosity measure ments of the different solvent ratios were represented in table 2. The change in viscosity with time for both solvent systems was shown in figure 4. It was found that by increas ing the acetic acid ratio up to 90%, the polymeric solution became more stable by time. This might be attributed to for mic acid high polarity (dielectric constant of 57.2ε o at 25 °C) in comparison to acetic acid polarity (dielectric constant of 6.6ε o at 25 °C) [18]. These results were in agreement with the results of De Vrieze and his coworkers [19] on their study of the instabilities of polyamide in pure formic acid. On the other hand, the pure acetic acid solution had a very low conductiv ity which leads to a highly viscous and stable solution, but it showed a difficulty in electrospinning. By combining FA and AA in adequate ratios, a solvent system with optimum stabil ity and viscosity was attained. As illustrated in table 2, by increasing the presence of acetic acid the conductivity decreases which increased the electrospinning difficulty. This was attributed to the low conductivity of acetic acid com pared to formic acid as stated above. Intermediate polarity is attained by combining acetic acid and formic acid of suitable ratio to get optimum electrospinning conditions.

Fiber morphology
The scanning electron microscopy images of the produced fibers from 12.5, 15.0, 17.5 and 20.0% PCL were represented in figure 5. The fibers produced from 12.5% PCL (image (a)) showed some beads and some drop lets. This may be due to electrospraying occurrence at low concentration instead of electrospinning due to the low vis cosity and the high surface tension of the solution [20]. At suitable concentration, smooth nanofibers can be obtained [21]. Higher concentrations (17.5 and 20.0% PCL) caused the production of helixshaped microribbons [22]. Moreover, faster solidification of the jet occurred at higher polymer con centration due to the presence of higher amounts of entan glements which have increased the difficulty of leaving the needle thereby causing thicker fibers.

Rheological measurements
The viscosity values of the different PCL concentrations were represented in table 3. As noticed from the table, as the concentration increases the mean fiber diameter increases due to the rise in viscosity as the amount of PCL increases. The viscosity and also the sta bility were increased as the added amount of PCL increases as shown in figure 6. This may be attributed to the increase in PCL amount which takes more time to be degraded. The least required PCL concentration to get nanofibers was found to be 15.0%. As presented in table 3, by increasing the concentration of PCL, the conductivity value decreases, which may be attributed to the decrease in the free solvent ions causing a slight reduction in the total conductivity.

Fiber morphology of applying voltage study
The scanning electron microscopy images of the produced fibers by using the applied voltage of 15.0, 17.5 and 20.0 kV were represented in figure 7. At 15.0 kV, the mean fiber diam eter was found to be 127.9 nm ± 55.83 nm and the repro ducibility of the fibers was the lowest. By increasing the applied voltage, usually fiber diameter reduction occurs due to the increased stretching of the electrospinning jet; how ever increasing voltage was reported also to have the reverse effect [22,23]. At 17.5 kV, the fibers seemed to be tangled and with a higher reproducibility even though with a higher fiber diameter (146.6 nm ± 55.72 nm). The mean fiber diameter for 20.0 kV was found to be 123.3 nm ± 36.73 nm. As observed, there was no specific trend as reported by Yördem and his coworkers [24] and also by Li and Wang [22]. However, by increasing the applied voltage the reproducibility increases.

Fiber morphology of tip to collector distance study
The scanning electron microscopy images of the produced fibers at tip to collector distances 10.0, 12.5, 15.0 cm were pre sented in figure 8.  plays a direct role on jet flight time and electric field strength. At 10.0 cm, the flight time was shortened and also the solvent evaporation time, and the electric field strength was increased, which results in the increase of bead formation [25]. At 12.5 cm, more time was provided for the fluid jet to stretch fully and for the solvent to evaporate completely. At 15.0 cm, the gap was increased so the collected fibers were partially dried before reaching the collector and fully stretched, and therefore fiber diameter was reduced; however the fibers reproducibility decreased. The optimum tip to collector dis tance was found to be 12.5 cm.

Conclusion
In the present study, a comparison between the electrospun fibers produced by formic acid/acetic acid (FA/AA) and formic acid/acetone (FA/A) solvent systems was studied. It was found that FA/AA produced much finer fibers than that produced by FA/A solvent system. FA/AA solvent system was chosen for optimum conditions study. The optimum solvent ratio, concentration, tip to collector distance and voltage were also investigated. It was found that the optimum conditions for PCL nanofibers electrospinning were for 70:30 FA/AA solvent ratio with 15.0% PCL concentration at 20.0 kV and a tip to collector distance of 12.5 cm. NanoPCL will be further used in biomedical applications.