DENG Linfeng(鄧臨風(fēng)), NI Jiaqian(倪賈芊), HU Xuefeng(胡雪峰), JING Yuanyuan(景媛媛), WANG Jilong(王霽龍)*, LIU Li(劉 力)*, MA Ying(馬 瑩)

1 Key Laboratory of Textile Science & Technology， Ministry of Education, College of Textiles, Donghua University, Shanghai 201620, China2 Zhejiang Zhongda Testing Technology Services Co., Ltd., Shaoxing 312030, China

Abstract: Electrospun sodium alginate (SA) fibers, which are always considered as a kind of biocompatible and non-toxic materials, have great potential application in the biomedical field due to high specific surface area and large porosity. However, in order to facilitate the electrospinning process, another polymer should be added into the SA solution. The effect of the added polymer of polyethylene oxide (PEO), SA, and ethanol on tuning the beaded structure of electrospun fibers was evaluated. Pure SA electrospun membrane with a beaded structure was prepared. The results show that PEO can facilitate the fabrication, but the mass fractions of SA and ethanol are positively correlated with the bead forming. When the mass fraction of ethanol in the solution was 15.0% (mass fractions of SA and PEO were 1.0% and 1.5%, respectively), the average diameter of the obtained beads was 824.80 nm, and the average length was 2.88 μm. Besides, the fibrous structure can be maintained even after the removal of PEO by ethanol. After removing PEO, the average diameter of the beads was reduced to 578.73 nm and the average length was reduced to 2.34 μm.

Key words: sodium alginate (SA); polyethylene oxide (PEO); electrospinning; beaded structure

Introduction

Sodium alginate (SA), which is extracted from natural brown algae, is a polyelectrolyte type random linear block copolymer with high charge density. It is formed by the 1, 4 glycosidic bonds ofβ-D-mannuronic acid (M units) andα-L-guluronic acid (G units)[1-2]. Owing to biocompatibility, low cost, and ease of production and functionalization, alginate has attracted tremendous attentions for its potential applications in biological medicine[3], textile[4-5], tissue engineering[6-7]and water purification[8]in recent years. Generally, nanofibers obtained by electrospinning have high specific surface area and large porosity[9-10], which are considered as a potential candidate in filtration[11], food packaging[12], energy storage system[13-14], wastewater treatment[15-16], and flexible electronic devices[17-19]. However, the electrospinning of pure alginate nanofibrous membrane is hard to be gained. One reason is that SA is a polyanionic electrolyte, which possesses too high electrical conductivity to form the Taylor cone with good efflux effect. In addition, the G units of the SA structure result in the rigid chains of negatively charged molecules, and hinder the efficient chain entanglement to draw fibers when being put into high voltage[20-22].

To achieve pure alginate nanofibrous membrane, lots of efforts have been devoted, including physical or chemical modification of SA[23-24], and the addition of non-toxic and biocompatible polymer (e.g.poly(vinyl alcohol)(PVA)[25-26], and polyethylene oxide (PEO)[27-28]). Considering the problems of process and energy consumption, PEO is more suitable for electrospinning with SA than PVA[29]. PEO, as a crystalline and water-soluble polymer, has low toxicity and can form hydrogen bonds with SA[30]. At the same time, the addition of polymer is a simple method to help electrospinning of SA. Gutierrez-Gonzalezetal.[31]fabricated an SA and PEO composite loaded with curcumin (CU) through electrospinning, and crosslinked with trifluoroacetic acid. The final product possessed a higher ultimate tensile stress(from (4.3±2.0) MPa to (15.1±2.0) MPa at CU mass fraction of 10%), which could have potential applications in filtering and tissue engineering. However, owing to high molecular weight and non-biological properties, the synthetic polymers cannot be eliminated or degradedinvivo, which results in the underlying damage of the original biological properties and hinders the application domain in tissue engineering[32]. Therefore, a removal procedure of polymer is necessary to achieve pure alginate nanofibrous membrane. Doderoetal.[22]prepared alginate-based membrane with ZnO nanoparticles via the procedure of electrospinning to construct potential wound healing patches. PEO was added during electrospinning, but was eventually removed before being used for wound patch. Additionally, Qietal.[33]added calcium chloride to alginate solution to further form calcium alginate microspheres wrapped model drug bovine serum protein (BSA). Then electrospun beaded nanofiber was successfully obtained and used as a carrier for drug delivery. To date, the efforts have been focused on the fabrication of pure alginate nanofibrous membrane or alginate-based materials, whereas the bead-on-string structure on the alginate nanofibrous membrane is rarely studied.

In this study, SA and PEO electrospun nanofibrous membrane (SA/PEO electrospun membrane for short), as well as pure SA electrospun membrane, are prepared through the procedure of electrospinning. Based on the system of SA, PEO and ethanol (SA/PEO/ethanol system for short), the structures of smooth and beaded nanofibrous membrane are finally achieved with the various mass fractions of solutions. The morphology of the generated electrospun nanofibrous membrane is evaluated through scanning electron microscopy (SEM), and the influence of the formation mechanism and morphology of the string fibers is also analyzed.

1 Experiments

1.1 Materials

SA (medium viscosity) and PEO (a molecular weight of 900 000) were purchased from Sigma Aldrich, United Kingdom, and used without further purification. Ethanol (analytical reagent, mass fraction of 95%) was purchased from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China. The deionized water was made by the deionized water machine in the laboratory.

1.2 Preparation of electrospinning solutions

In order to prepare SA aqueous solutions with a total volume of 10 mL, 0.10 g or 0.15 g SA powder and a certain amount of distilled water were poured into a small sample flask. The SA solutions were then stirred at room temperature for 24 h until the solutions were homogeneous. Next, various amounts of PEO (0.15, 0.20, and 0.30 g) were measured and added to the obtained SA solutions. The solutions were stirred at room temperature with magnetic stirring for another 24 h. If ethanol was added to the solution, the mass ratio of ethanol to water in the solution was changed in order to control the size of the beads in the spinning process. Furthermore, ethanol was universally added in the end[34].

1.3 Electrospinning of SA/PEO solutions

Different kinds of SA/PEO solutions were placed in a syringe (5 mL) and fixed in a spinning extruder pump using a needle with the inner diameter of 0.57 mm. The positive lead of the high voltage power supply was connected to the needle through a small clamp, and the lead of the negative pressure power supply was connected to the receiving roller. The receiving roller was adhered with aluminum foil as the receiving device. In addition, the receiving distance was set to be 10 cm and the flow rates of the solution were 0.40-0.65 mL/h. The positive voltage was kept at 12.5 kV, while the negative voltage was -0.9 kV.

1.4 Removal of PEO

Firstly, the SA/PEO electrospun membrane, which was obtained by electrospinning, was immersed in hot ethanol at 70 ℃ for 24 h to adequately remove the existence of PEO. Then the pure SA electrospun membrane was successfully prepared. Finally, it was washed several times in deionized water and dried in an oven at 50 ℃ for 12 h.

1.5 Morphology analysis

The morphology of the electrospun membrane was examined using SEM (SU-8010, Japan HITACHI Company, Japan) at an accelerating voltage of 5 kV. Image analysis software (Image J, Rawak Software Inc., Stuttgart, Germany) was used to determine the average diameter of the nanofibers by analyzing approximately 100 points, while the average diameter and the average length of the beads were determined by measuring 30 points, respectively.

1.6 Spectroscopic analysis

The infrared spectra of the membrane were analyzed using a Fourier transformed infrared (FTIR) spectrometer (Nicolet 6700, America Thermo Fisher Company, USA). Samples were scanned 32 times in the range from 600 cm-1to 4 000 cm-1at a resolution of 4 cm-1.

1.7 Conductivity analysis

The electrical conductivity of various SA/PEO solutions was measured by a conductivity meter (Five Easy Plus, FE38, Mettler-Toledo Conductivity Meter, Shanghai, China). Each solution was measured at different locations for three times to take the average conductivity.

1.8 Surface tension analysis

The surface tension of the prepared SA/PEO solutions was determined by a surface tensiometer (DCA T11, Germany Dataphysics Instrument GmbH, Germany).

2 Results and Discussion

Due to difficulties to produce SA electrospun membrane, it was proposed to electrospin the SA/PEO solution giving a bead-on-string structure. With the process of electrospinning and heating, pure SA electrospun membrane could be obtained by adjusting various parameters of the solvent in spinning solution. The whole procedure of fabricating pure SA electrospun membrane was shown in Fig. 1.

Fig. 1 Fabricating procedure of SA electrospun membrane: (a) preparation of SA/PEO spinning solution; (b) basic structure of SA and PEO; (c) process of electrospinning; (d) obtained SA/PEO electrospun membrane; (e) removal process of PEO; (f) final SA electrospun membrane

Figure 2 showed that the morphology of the electrospun membranes with different mass fractions of ethanol. From Figs. 2(a), 2(d) and 2(g), the distribution of SA/PEO electrospun membrane without ethanol was regular and the fiber diameter was uniform; with the addition of ethanol as a cosolvent in the electrospinning solution, the beaded structure began to appear in the nanofibrous membrane; when the ethanol mass fraction was 10.0%, the average diameter of the beads was up to 661.13 nm and the average length was 2.06 μm by the measurement of Image J software. Moreover, as shown in Figs. 2(c), 2(f) and 2(i), it was not difficult in finding that the increase of the mass fraction of ethanol (15.0%) would be conducive to increasing the number of beads. At this time, the average diameter of the beads was up to 824.80 nm that was larger than the bead diameter for ethanol mass fraction of 10.0%. Besides, the average length of beads for ethanol mass fraction of 15.0% was 2.88 μm, which was larger than that for ethanol mass fraction of 10.0%.

Fig. 2 SEM images of SA/PEO electrospun membrane (SA mass fraction of 1.0% and PEO mass fraction of 1.5%) with different ethanol mass fractions: (a)-(c) 0; (d)-(f) 10.0%; (g)-(i) 15.0% is the average length)

Firstly, owing to low surface tension, the existence of ethanol would reduce the surface tension of the whole spinning solution to some extent, thus declining the production of beads during the process of electrospinning. When adding SA in the spinning solution, the surface tension increased. At the same time, the larger portion of SA played a more significant role in increasing the surface tension than ethanol, which contributed to the probability of beaded electrospun membrane.

Secondly, with the addition of the ethanol to the spinning solution, the low dielectric constant of ethanol prevented the ionization of COO— in the macromolecular structure of SA. Consequently, it would lead to the lower conductivity of the spinning solution, as shown in Fig. 3, and the descend of the charge density on the jet surface, as well as reducing the effect of the charge repulsion on the jet surface. Therefore, it was no denying that the stretching of solution was reduced and the fiber became more refined. Meantime, the Rayleigh instability movement of the jet, which is an axisymmetric motion driven by surface tension, occupies a dominant position under the condition of the low conductivity[35]. At the moment, the surface tension of the SA/PEO/ethanol system accelerated the larger Rayleigh instability movement, thereby gaining the production of homogeneous beads on the SA/PEO electrospun membrane.

Fig. 3 Conductivity and surface tension results of various spinning solutions with different ethanol mass fractions

It was not difficult to see from Figs. 4(a) and 4(b) that with the increase of SA, the number of beads in the electrospun membrane increased significantly. Also, as shown in Fig. 5, conductivity and surface tension increased with more addition of SA. When the mass fraction of ethanol was 15.0%, the continuous and uniform morphology of nanofibers appeared, and there was random distribution of beads on the surface. What can be used to explain this is that SA, as an anionic polyelectrolyte, has increased the charge density in the ejected jets and then led to thinner formation of electrospun membrane[36]. Moreover, the ionic bonds contained in the polyelectrolyte will produce strong repulsive forces and further reduce the effect of external electric field, preventing the formation of continuous straight fibers to some extent[21]. As a consequence, the morphology of the membrane was changed from homogeneous fibers to beaded ones by the enhancement of SA content.

Fig. 4 SEM images of SA/PEO electrospun membrane (PEO mass fraction of 1.5% and ethanol mass fraction of 15.0%) with different SA mass fractions: (a) 1.0%; (b) 1.5%

Fig. 5 Conductivity and surface tension of two solutions

The challenges of alginate electrospinning can be ascribed to its polyelectrolyte nature and chain conformation characteristics. Additionally, the —OH groups contained in the structure of PEO are bound to create a strong hydrogen bond with the COO— groups of SA, resulting in flexible entanglements to conduct spinning process. Moreover, it makes the electrospinning of SA possibly and expands the potential application of SA in the electrospinning. From Fig. 6, the electrospun membrane with different mass fractions of PEO exhibited good nanofibrous morphology without the generation of beads, and the diameters of the nanofibers were relatively uniform. Meantime, with the increasing mass fraction of PEO, the average diameter of the nanofibers was distributed in an upward trend from 145.85 nm to 203.85 nm.

Fig. 6 SEM images of SA/PEO electrospun membrane (SA mass fraction of 1.0%) with different PEO mass fractions: (a) 1.5%; (b) 2.0%; (c) 3.0%

As a representative carrier polymer, PEO has been widely used to enhance the spinnability of polysaccharide or pectin solution and to ensure that there are enough flexible entanglements of molecular chains to assist the forming of electrospun membrane. Nevertheless, the synthetic polymer (PEO) cannot be effectively eliminated and naturally degraded in organisms with the possibility of damaging the original characteristics of organisms, which greatly imposes restrictions on the potential application in the field of medical biology. Therefore, it is highly desirable to eliminate the high polymer of PEO from the SA/PEO electrospun membrane to gain the relatively pure SA electrospun membrane.

In this study, hot ethanol solution (70 ℃) was used to soak the SA/PEO electrospun membrane for 24 h to fully remove PEO. It was not difficult to observe from Fig. 7(a) that after immersing in hot ethanol, the morphology of the sample without ethanol was presented nanofibers with uniform diameter distribution. The results in Fig. 7(b) showed that the size of the beads after eliminating PEO could also be maintained, which indicated that it was effective to obtain pure alginate electrospun membrane. PEO is insoluble in ethanol at room temperature whereas the chemical structures of PEO [CH2CH2O] and ethanol molecules (H—CH2CH2—OH) are similar. Therefore, when the solution temperature is higher than the melting temperature of PEO, ethanol can be used as a good solvent to dissolve PEO.

Fig. 7 SEM images of pure SA electrospun membrane after the removal of PEO (SA mass fraction of 1.0% and original PEO mass fraction of 1.5%) with different mass fractions of ethanol: (a) 0； (b) 15.0%

Figure 8(a) reports the FTIR spectra collected in an attenuated total reflectance(ATR) mode for the SA/PEO electrospun membrane (SA mass fraction of 1.0% and orginal PEO mass fraction of 1.5%) and pure SA electrospun membrane after removing PEO compared with SA and PEO powders. The spectral profiles of the SA/PEO electrospun membrane strongly resemble those of PEO powder with slight difference probably due to the existence of SA. The large unstructed bands in the 3 280 cm-1of SA powder and 3 365 cm-1of pure SA electrospun membrane were attributed to the stretching vibrations of hydroxyl groups. The bands at 1 597 cm-1and 1 603 cm-1were assigned to asymmetric stretching vibrations of the carboxylic groups, while other bands at 1 409 cm-1and 1 413 cm-1were assigned to symmetric stretching vibrations of the carboxylic groups, respectively. Additionally, the signals at 1 100 cm-1and 1 098 cm-1corresponded to C—O—C vibrations whereas those falling at 962 cm-1were attributable to C—O vibrations. With viewing the characteristic peaks of eletrospun membrane and powders, it was not hard to observe that the vibration peaks of pure SA electrospun membrane after removing PEO were basically consistent with those of pure SA powder. And there were no peaks of PEO, fully indicating that hot ethanol (70 ℃) was effective in removing PEO. Also, this method would contribute to obtaining pure SA electrospun membrane to achieve favorable biocompatibility and biodegradability of alginate applications.

Figure 8(b) exhibits that the FTIR spectra collected in the ATR mode for the SA/PEO electrospun membrane (SA mass fraction of 1.0%, original PEO mass fraction of 1.5%, and ethanol mass fraction of 15.0%) and pure SA electrospun membrane after removing PEO compared with SA and PEO powders. The large unstructed bands at 3 280 cm-1of SA powder and 3 365 cm-1of pure SA electrospun membrane were attributed to the stretching vibrations of hydroxyl groups. The bands at 1 597 cm-1and 1 600 cm-1were assigned to asymmetric stretching vibrations of the carboxylic groups, while other bands at 1 409 cm-1and 1 411 cm-1were assigned to symmetric stretching vibrations of the carboxylic groups, respectively. Meantime, due to the existence of ethanol, there were still some differences between the SA/PEO electrospun membrane and PEO powder. Indeed, PEO could be removed after immersion in hot ethanol. As shown in Fig.7(b), the average diameter and the average length of the final beads were 578.73 nm and 2.34 μm, respectively. Also, the obtained pure SA electrospun membrane had good biocompatibility.

Fig. 8 FTIR spectra results of pure SA electrospun membrane after removal of PEO (SA mass fraction of 1.0% and original PEO mass fraction of 1.5%) with different mass fractions of ethanol: (a) 0; (b) 15.0%

3 Conclusions

Pure SA nanofibrous membrane was successfully prepared via electrospinning technique. The addition of PEO was aimed at assisting the electrospinning of alginate, and with the complete removal of PEO, pure SA electrospun membrane could be obtained. In the presence of alginate and ethanol, the electrospun membrane was able to form beaded structure. Meantime, the size of beads increased with the elevation of ethanol, and the increasing amount of the alginate led to the increasing number of beads. Moreover, the beaded nanofibrous membrane of alginate is low-cost, biocompatible and harmless to human body and the environment without the addition of any synthetic polymer. In the future, considering the factors of process parameters on the morphology of beads, the size and the density of the beads can be further controlled, so as to expand the range of drug-loading particles. In addition, with the enhancement of drug delivery performance and biocompatibilityinvivo, the beaded electrospun nanofibrous membrane is expected to achieve effective application in the field of tissue engineering and drug release.