Temperature effects on electrospun chitosan nanofibers

Abstract Effects of the temperature of chitosan (CS) solutions as well as the temperature of the chamber on an electrospinning process were investigated. CS with a low molecular weight was dissolved in the solvent of trifluoroacetic acid/dichloromethane (70/30 v/v) at a concentration of 80 mg/mL for electrospinning. Both CS solution and chamber temperatures strongly affected the morphology of electrospun CS nanofibers. At the solution temperature and chamber temperature of 32°C, uniform CS nanofibers with an average diameter of 200 nm could be obtained. Although the chamber temperature is generally regarded as an unimportant parameter in the electrospinning of polymers, the experimental results demonstrated its critical effect on the electrospinning of CS.

Besides the above advantages of CS, nanofibrous CS substrates can provide a high surface area for adsorption and cell attachment [1,6]. Thus, CS nanofibers should have a high potential use for tissue-engineering scaffolds as well as adsorption processes [1,2,7]. However, electrospinning of CS is limited due to its high viscosity, the limitation of solvents, and the positive charges of amino groups [8]. Although there are a few reports about the electrospinning of pure CS, important operational parameters for the electrospinning of CS have not been investigated thoroughly. Ohkawa et al. (2004) reported the first successful preparation of pure CS fibers by the electrospinning technique. By using trifluoroacetic acid (TFA) as the solvent, homogeneous CS nanofibers were successfully prepared [9]. High volatility of TFA plays a crucial role in the formation of uniform nanofibers. Moreover, TFA can block the positive charges of the amino groups on CS, which can decrease the electrostatic forces and enable the production of continuous fibers [9,10]. Therefore, the effectiveness of the TFA fostered further studies on electrospinning CS with various molecular weights where TFA was utilized as the solvent [11,12].
Electrospinning can create very fine fibers from a liquid by using an electrostatic force. When the electrical field is applied, charges on the surface of a polymer solution repulse each other. Meanwhile, surface tension causes forces opposite to the electrical force. With counteraction between mutual repulsive force between charges and the force from surface tension, the liquid is bended to form an electrospun jet. The eruption of the liquid has a conical shape, called as the Taylor cone [13][14][15][16]. Then, the jet is continuously elongated, and the solvent is also evaporated to form fibers if the entanglement force in the liquid is high enough to prevent the breaking of the stream. Finally, fibers are deposited on the collector. Electrospinning is a simple process to produce micro-and nanofibers. However, the effects of parameters are very complicated [13,17]. The electrospinning parameters are usually divided into three groups: the properties of a polymer solution (e.g., polymer concentration, polymer molecular weight, the type of solvent); operational parameters (e.g., the applied voltage, the distance between the needle tip and collector, feeding rate); and ambient conditions (e.g., temperature, humidity, velocity of air chamber) [13,17,18].
While various studies focused on polymer solution and operational parameters, ambient parameters are generally considered to contribute an insignificant effect to the electrospinning process. However, it should be noted that an increase in the temperature of a polymer solution might lead to a decrease in the viscosity of that solution and an increase in the evaporation of the solvent [19]. Thus, an increase in temperature of a polymer solution could lead to a decrease in diameters of electrospun fibers. Therefore, there are several studies on the effects of the polymer solution temperature on electrospinning. The results show that increasing the temperature is often beneficial to the process of electrospinning [19][20][21][22]. In addition, melting electrospinning is also an attractive issue because there is no need to use solvents for electrospinning [23][24][25].
Although uniform CS nanofibers could be prepared by using the TFA/dichloromethane (DCM) as the solvent, the temperature effect of the CS solution has not been investigated. Also, it is questionable whether the temperature of the electrospinning chamber might have a significant effect on the evaporation of the solvent. To address this knowledge gap, this research work investigated the effects of both solution temperature and chamber temperature on electrospun CS nanofibers.
Rigorous experimentation on the impact of various types of solvents on the nanofiber morphology was also conducted to reconfirm the effectiveness of TFA/DCM for the successful electrospinning of pure CS nanofibers.

Materials
Ascorbic acid, glycolic acid, malic acid, succinic acid, TFA, ethanol, hexane, tetrahydrofuran (THF), and CS with a low molecular weight were purchased from Sigma-Aldrich. DCM was obtained from TEDIA.

Electrospinning of CS
In this study, we developed a customized electrospinning setup, as shown in Figure 1. There are three main components in the electrospinning equipment: a syringe pump (KD Scientific, KDS 100) with a metallic needle (0.65 × 32 mm), where the temperature of the solution is controlled; a high voltage generator; and a collector covered by an aluminum foil.
The solution temperature was controlled by surrounding the needle and the syringe with a tube that allowed a continuous flow of water from a temperaturecontrolled water bath (as illustrated in Figure 1, Section I). An electric heater attached on both sides of the chamber was used to warm up the chamber to the desired chamber temperature measured by a thermocouple that was placed close to the workspace of the collector (as illustrated in Figure 1).
In this study, CS solutions were prepared for electrospinning using various solvents with different levels of volatility. First, low-volatility acids (i.e., ascorbic acid, glycolic acid, malic acid, succinic acid) were utilized to produce CS solutions of 2-5% concentration for electrospinning. Second, acetic acid, a moderate-volatility acid, was also used to produce CS solutions of 5-7% concentration for electrospinning. Cosolvents (i.e., ethanol, hexane, THF) were also added into the solution to analyze their effect on electrospinning products. Finally, CS was dissolved by strong and high-volatility TFA with DCM as the co-solvent, as described by the previous report for preparing pure CS nanofibers [9]. CS was dissolved in TFA/DCM (70/30 v/v) at a concentration of 80 mg/mL. After stirring for 12 h, the CS solution was placed in a 3 mL syringe for electrospinning. The distance and the applied voltage between the tip and the collector were 120 mm and 17 kV, respectively. The flow rate of CS solution was fixed at 0.2 mL/h. The electrospinning experiments were carried out at a humidity of 70-75% for 45 min. To study the temperature effects of the solution, the solution temperatures of 20°C, 22°C, 27°C, and 32°C were observed under experimentation after the chamber temperature had been fixed at 32°C. To investigate the chamber temperature, the experiments were conducted at the chamber temperatures of 24°C, 29°C, 32°C, and 37°C after the solution temperature had been controlled at 32°C.

Characterizations of electrospun CS nanofibers
Surface morphology of the CS nanofibers was observed by a scanning electron microscope (SEM, JSM-6390LV, JEOL, Japan) at an accelerating voltage of 20 kV after gold coating. From the SEM image results, the diameters of CS fibers and the distribution of fiber diameters were analyzed by ImageJ software. A hundred fibers were randomly selected from different areas of a SEM image to determine the mean diameter and its standard deviation of the obtained nanofibers.

Effects of the solvent
Besides TFA, various solvents for CS including lowvolatility acids such as ascorbic acid, glycolic acid, malic acid, and succinic acid; and a moderate-volatility acid (i.e., acetic acid and its mixtures with various co-solvents) were experimented in this study. Figure 2 shows the SEM images of electrospinning CS using solvents of the former type. No uniform structure could be observed. It was known that TFA could change the viscosity of a CS solution and the interaction between the amino groups of CS [9], resulting in the electrospinnability of pure CS. For ascorbic acid, succinic acid, and malic acid, they increased the viscosity of a CS solution, revealing the enhancement in the entanglement force between the polymer chains of CS [26]. Thus, no homogeneous and continuous fibers could be formed by using these solvents. Figure 3 shows the SEM images of electrospun CS with acetic acid and the mixture of acetic acid with cosolvents including hexane, ethanol, and THF. These cosolvents were chosen due to their low surface tension, low dielectric constant, and high volatility ( Table 1). It was found that the bead-in-string morphology existed, and the homogeneity of CS nanofibers was not improved using different co-solvents. These factors agreed well with the previous reports in ref. [9,27], which supported the use of TFA as a high-volatility solvent to suppress the positive charges of CS for the successful electrospinning of pure CS. Figure 4 shows the SEM images of the electrospun fibers when the solution temperatures were varied from 20°C, 22°C, 27°C, and 32°C. Remarkably, morphology change from bead-connected fibers to uniform fibers was  observed, following the increase in solution temperature. This finding could be very useful for the formation of uniform electrospun fibers and was consistent with previous research works on the electrospinning of CS/ poly(acrylamide) [20], CS/polyethylene oxide [19], and nylon 6 [21].

Effects of the solution temperature
Because the high viscosity of CS solution may limit the electrospinning process, a high concentration of CS with a low molecular weight was used as a trade-off between lowering viscosity (i.e., using low-molecular weight CS) and allowing the successful electrospinning of CS nanofibers (i.e., increasing concentration to provide enough CS material). Therefore, an increase in the solution temperature might lead to a decrease in the viscosity of CS solution. As a consequence, the entanglement force could be reduced and the electrospinning process could get accelerated to produce uniform CS nanofibers.
The solution temperature increase could also result in an increase in chain mobility and repulsive interaction among polymer chains. Thus, increasing solution temperature could partially promote the formation of fibers and limit the formation of beads. These tendencies could have facilitated the formation of uniform electrospun fibers. Figure 5 shows the SEM images of the electrospun CS fibers at various chamber temperatures (24°C, 29°C, 32°C, and 37°C). Uniform CS nanofibers were obtained at the chamber temperature of 32°C. An increase in the chamber temperature could have resulted in a higher evaporation rate of the solvent; thus, the electrical forces could be reduced during the electrospinning process. However, at a higher chamber temperature (i.e., 37°C), the electrospinning jet was solidified, which resulted in the production of inhomogeneous fibers. Therefore, chamber temperature should be considered as a crucial electrospinning parameter for obtaining uniform CS nanofibers. Figure 6 shows the distribution of the CS nanofiber diameters obtained under the optimal temperature conditions. The diameter of the obtained CS nanofibers had a mean value of about 200 nm with a standard deviation of 86 nm.

Conclusion
Uniform nanofibers with an average diameter of 200 nm were successfully obtained from pure CS by the electrospinning technique. The TFA/DCM (70/30 v/v) solvents were the best choice for the electrospinning process. The temperature of the CS solution and the chamber had significant effects on the morphology of CS nanofibers. The proper temperatures of the solution and the chamber were 32°C for preparing pure CS nanofibers, which would be a potential factor for various applications in biomaterials, adsorbents, and carriers.