Correlation of Chitosan's Rheological Properties and Its Ability to

Biomacromolecules 2008, 9, 2947–2953

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Correlation of Chitosan’s Rheological Properties and Its Ability to Electrospin Rebecca R. Klossner,† Hailey A. Queen,‡ Andrew J. Coughlin,‡,§ and Wendy E. Krause*,†,‡ Fiber and Polymer Science Program and Department of Textile Engineering, Chemistry and Science, North Carolina State University, 2401 Research Drive, Campus Box 8301, Raleigh, North Carolina 27695 Received July 3, 2008; Revised Manuscript Received August 12, 2008

Chitosan-based, defect-free nanofibers with average diameters ranging from 62 ( 9 nm to 129 ( 16 nm were fabricated via electrospinning blended solutions of chitosan and polyethylene oxide (PEO). Several solution parameters such as acetic acid concentration, polymer concentration, and polymer molecular weight were investigated to optimize fiber consistency and diameter. These parameters were evaluated using the rheological properties of the solutions as well as images produced by scanning electron microscopy (SEM) of the electrospun nanofibers. Generally, SEM imaging demonstrated that as total polymer concentration (chitosan + PEO) increased, the number of beads decreased, and as chitosan concentration increased, fiber diameter decreased. Chitosan-PEO solutions phase separate over time; as a result, blended solutions were able to be electrospun with the weakest electric field and the least amount of complications when solutions were electrospun within 24 h of initially being blended. The addition of NaCl stabilized these solutions and increased the time the blended solutions could be stored before electrospinning. Pure chitosan nanofibers with high degrees of deacetylation (about 80%) were unable to be produced. When attempting to electrospin highly deacetylated chitosan from aqueous acetic acid at concentrations above the entanglement concentration, the electric field was insufficient to overcome the combined effect of the surface tension and viscosity of the solution. Therefore, the degree of deacetylation is an extremely important parameter to consider when attempting to electrospin chitosan.

Introduction Electrospinning has been an area of growing interest in the medical community because of its ability to produce nanosized fibers with high surface area that mimic the extracellular matrix1 and can be used in a variety of applications such as scaffolds for tissue engineering,2-6 drug delivery devices,7-9 multifunctional membranes,10 artificial organs,11,12 wound dressings,12-15 and vascular grafts.9,11,12,16 Generally, most electrospinning attempts focus primarily on synthetic polymer systems. However, natural biopolymers, such as chitosan, have attracted attention because of their excellent biocompatibility and biodegradability.1 Chitosan has several biological properties that make it an attractive material for use in medical applications such as biodegradability, lack of toxicity, antifungal effects, wound healing acceleration, hemostatic nature, and immune system stimulation.5,17-19 Chitosan is the deacetylated derivative of chitin, which the second most abundant polysaccharide in the world, after cellulose. Chitosan is typically produced from chitin through the use of chemical or enzymatic treatments of the shells of Arthropoda (shrimp or crab) secured from the waste products of the crabbing and shrimping industries.17 Chitosan has proven challenging to electrospin, as have many other naturally occurring biopolymers. First, chitosan’s rigid D-glucosamine repeat unit, high crystallinity, and ability to hydrogen bond lead to poor solubility in common organic solvents.20 McKee et al. found that to electrospin defect-free * To whom correspondence should be addressed. E-mail: wekrause@ ncsu.edu. † Fiber and Polymer Science Program. ‡ Department of Textile Engineering, Chemistry and Science. § Present address: Department of Bioengineering, Rice University, Houston, TX 77005.

fibers from polymer solutions the concentration of the polymer must be at least 2 to 2.5 times the entanglement concentration.21 The entanglement concentration, ce, is the boundary between the semidilute unentangled regime (where polymer chains overlap one another but are not entangled) and the semidilute entangled regime (where the polymer chains significantly overlap one another and topologically constrain each other’s motion). However, chitosan solutions at these concentrations are often difficult to electrospin because of the high viscosity in solution.20,22-30 Therefore, the rheological behavior of the polymer solution plays a crucial role in the electrospinnability for any given sample. With chitosan solutions, even moderate concentrations become too viscous to overcome the electric field and cannot be successfully electrospun. Additionally, chitosan is a cationic biopolymer that also affects the rheology of the solutions. McKee et al. show that while neutral polymers often form beaded nanofibers at the entanglement concentration (ce), salt-free polyelectrolyte solutions of poly((2-dimethylamino)ethyl methacrylate) (PDMAEMA) did not form fibers until eight times the ce.21 A chitosan solution of moderate molecular weight (148000 g/mol) that is eight times the ce would have a viscosity of over 170000 P, which would be nearly impossible to electrospin using a laboratory size setup. With a typical laboratory electrospinning setup, the viscosity of the solution must be within a certain window for nanofibers to form successfully. Above the upper threshold the solution becomes too viscous and fiber formation is hindered because the electric field is not strong enough to overcome the viscosity of the solution. Below the lower limit, the polymer chains are not entangled so fiber formation is not possible and polymer beads often are created. Therefore, several groups have blended chitosan with other polymers in an attempt to improve the electrospinnability of the solutions.1,28,30,31

10.1021/bm800738u CCC: $40.75  2008 American Chemical Society Published on Web 09/12/2008

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Biomacromolecules, Vol. 9, No. 10, 2008

Since 2004, reports of electrospinning chitosan in blended solutions with varying degrees of success have been made.22-24,26,27,29,31-35 Other studies have reported nearly defect free nanofibers with slightly larger fiber diameters using poly(ethylene oxide) (PEO)/chitosan blends.23,27,28,31,36 The successful electrospinning of pure chitosan has been reported by several groups and has recently been summarized by Schiffman et al.31 Trifluoroacetic acid (TFA) is most often used as the solvent to successfully electrospin chitosan. However, two groups have fabricated chitosan nanofibers using a solvent system of aqueous acetic acid. The first group, Geng et al. used a solvent system of 90% acetic acid and a 7 wt % concentration of chitosan (molecular weight of 106000 g/mol) with a 54% degree of deacetylation (DD).24 The second group, De Vrieze et al. also used a system with 90% acetic acid, however, they were able to fabricate fibers using only a 3 wt % solution of chitosan (molecular weight ranging from 190000 to 310000 g/mol) with a DD of 75-85%.35 We attempted to electrospin pure chitosan (molecular weight 148000 g/mol) with a DD of 75-85% in a concentrated acetic acid solvent system. Additionally, chitosan-PEO blends were electrospun into defect free nanofibers with diameters in the range of 62 to 129 nm. Finally, time studies were carried out to determine the effect on the zero shear rate viscosity (η0), and the electrospinnability of chitosan blended solutions.

Experimental Section Materials. The materials used in these experiments include a solvent system of glacial acetic acid, obtained from VWR International, and deionized water; while the solutes consisted of poly(ethylene oxide) (PEO) and three molecular weights of chitosan. The PEO has a molecular weight of 900000 g/mol and was obtained from Scientific Polymer Products, Inc. The chitosan samples were obtained from Fluka and have molecular weights of 600000, 400000, and 148000 g/mol. The average molecular weight of the lowest molecular weight chitosan sample was confirmed using intrinsic viscosity and the Mark-Houwink equation, [η] ) KMa, where K ) 1.81 × 10-3 mL/g and a ) 0.93.37 The average molecular weight was determined to be 148000 g/mol and the intrinsic viscosity [η] was measured in a mixture of 0.1 M acetic acid and 0.2 M sodium chloride at 25 °C.38,39 Additionally, each of the three chitosan samples have a degree of deacetylation of 75-85%. All reagents and polymers were used as received. Electrospinning Apparatus. The electrospinning apparatus was set up horizontally and included a 10 mL syringe with luer-lock connections, a 4 in. 20 gauge blunt tip needle (Becton, Dickinson and Company), a programmable syringe pump (NE-1000 New Era Pump Systems Inc.), and a high-voltage power supply (Glassman FC60R2 with a positive polarity). Typical operating parameters used in this study are flow rates between 1 and 10 µm/min and voltages between 7.5 and 10.5 kV. Additionally, the experiments required that the system include a digitally controlled polymer flow, a wide range of electric field strengths, an adjustable needle to collector distance (usually 10 cm), and a single collector setup. The single collector plate allowed for the fibers to be collected in a random array. The fibers were collected on aluminum foil, which was placed over the collector plate for uncomplicated sample removal.40 Methods. Various ratios of acetic acid in deionized water were used as the solvent system, ranging from 10 to 90%. Various weight percents of the three different molecular weight samples of chitosan, ranging from 0.5 to 8%, as well as blends of PEO and chitosan were investigated. After the solvent and the solute were added, the complete solution was stirred on a magnetic stir plate for a period of 24 to 72 h until the solution was homogeneous. The solutions were then stored in a sealed container at room temperature.

Klossner et al. Table 1. Chitosan-PEO Blended Solutions

sample A B C D E F G H

CSa conc. 2 2 2 2 3 4 5 5

wt wt wt wt wt wt wt wt

% % % % % % % %

AAa ratio of resulting conc. CS solution polymer of CS to PEO resulting conc. b solution solution AA conc. (CS + PEO) 90% 90% 80% 80% 80% 80% 80% 80%

50:50 40:60 50:50 40:60 40:60 40:60 40:60 50:50

45% 36% 40% 32% 32% 32% 32% 40%

2.5 2.6 2.5 2.6 3.0 3.4 3.8 4.0


% % % % % % % %

a AA and CS represent acetic acid and chitosan, respectively. b All PEO solutions were 3 wt % in deionized water. The molecular weight of the chitosan and PEO was 148000 and 900000, respectively.

Blended solutions were prepared by making separate 148000 g/mol chitosan-acetic acid solutions of varied concentration and a 3 wt % PEO-water solution and then mixing the two solutions together in various chitosan/PEO solution ratios (see Table 1). The combined solutions were then stirred on a magnetic stir plate for at least two hours to ensure adequate mixing. The solutions were stored in a sealed container at room temperature. Electrospinning of Chitosan and Blended Solutions. Electrospinning of the solutions occurred at room temperature. The fully dissolved solutions were drawn into the syringe and a 20 gauge needle was attached. All the air is manually removed from the needle by pushing the polymer solution through the syringe until it emerges at the end of the needle. The syringe is then secured in the syringe pump and the flow rate on the pump is set between 1 and 15 µL/min. The flow rate must be balanced with the electric field and viscosity of the solution intended for electrospinning. Increasing the electric field strength will draw the polymer from the needle, requiring a higher flow rate. If the flow rate is too high, then the polymer jet is interrupted and the polymer solution drips from the needle. Therefore, the flow rate was determined on a solution to solution basis. Precautions are taken to ensure the electrospinning system is grounded. The high voltage supply is connected to the end of the needle farthest away from the syringe pump to prevent interference with the pump from the electric field. The high voltage supply is then powered on and when a droplet of polymer solution appears at the end of the needle the voltage is turned up until a Taylor cone becomes visible and a liquid jet of polymer is seen being pulled toward the grounded collector plate, which was typically between 7 and 25 kV. The electrospinning process is then allowed to continue from 30 min to 2 h until a solid white sample is visible on the aluminum foil covered collector plate. The aluminum foil is a generic brand that is simply pulled over the edges of the circular collection plate and is easily removed after spinning. Characterization. The samples were characterized using various methods. First, rheological studies are used to determine the zero shear rate viscosity (η0) of the solutions used in electrospinning because it has a dramatic effect on the ability to form nanofibers. Additionally, scanning electron microscopy (SEM) is used to examine the quality of the electrospun material and determine the diameter of the resulting fibers. Rheology. The StressTech HR (ATS RheoSystems, Bordentown, NJ), a stress controlled rheometer, was used to obtain the zero-shear rate viscosity (η0) of all of the polymer solutions. A 50 mm parallel plate was used to collect the viscosity data. The gap used for all solutions was 0.300 mm at a temperature of 25 ( 0.1 °C. Additionally, to reach low shear rates with a low viscosity sample, a custom-made, double-gap, concentric cylinder geometry was used. Generally, the concentric cylinder geometry is used with polymer solutions below 1000 cP and the parallel plates are used for solutions above that value. The outer radius of the bob measures 26.22 mm, with an inner radius of 21.60 mm. A volume of 2.83 cm3 is required for accurate measurements.

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Figure 1. Typical micrograph of “electrospun” chitosan in acetic acid/ water (this example: MW) 148000 g/mol, conc. ) 3 wt %, and acetic acid conc. ) 45%).

This fixture allowed for precise rheology measurements at low concentrations of chitosan. The chitosan solutions were measured within two weeks of preparation, while the chitosan/PEO solutions were measured as a function of time. Scanning Electron Microscopy. From the collection material (aluminum foil), samples of approximately 1 × 1 cm were cut. The samples were then coated with a layer of gold about 100 Å thick using a gold-sputter machine to reduce charge interruptions. Following coating the samples were mounted in the JEOL 6400F field emission SEM. The samples were then focused, and viewed at magnifications between 5000-40000 times their original sizes. Those images were then used to visually evaluate fiber diameter and consistency. The average fiber diameter was determined using Scion Imaging software in conjunction with the SEM image. The software measures the size of certain features in the SEM image. Ten different fiber diameters were determined and averaged to find the fiber diameter reported for each of the resulting electrospun mats. The 95% confidence limits of the mean were also calculated and reported with each average fiber diameter.

Results and Discussion Although chitosan has proven to be challenging to electrospin, it has many exceptional biological properties which would be beneficial in nanofibrous mats for use in biomedical applications such as tissue engineering. Therefore, we first attempted to electrospin pure chitosan fibers to capitalize on these unique characteristics. The first aspect of the polymer that was considered was the molecular weight which has a profound effect on the viscosity of a solution and, thus, the ability to electrospin that solution. Three molecular weights of chitosan were used in these series of experiments to determine which degree of molecular weight would be optimal. Solutions of 600000, 400000 and 148000 g/mol chitosan (each with a degree of deacetylation of 75-85%) were made in concentrations from 1 to 8 wt % in increments of 1 wt %. Electrospinning was attempted with all the solutions without the production of nanofibers. The lowest molecular weight chitosan sample had the lowest solution viscosity, was considered the most promising, and therefore, was the only sample investigated further. An acetic acid-water blend was found to be an appropriate solvent that would not leave a toxic residual in the fibers. The concentration of acetic acid was varied in increments of 10% to fully dissolve the chitosan. The viscosity of the solutions was roughly equivalent over the entire range (10-90%) of acetic acid concentration, in agreement with Geng et al.24 Attempts to electrospin these solutions were either unsuccessful or ended with beaded structures instead of nanofibers (Figure 1). Electrospinning chitosan with high degrees of deacetylation is

Figure 2. Plot of specific viscosity (ηsp) versus concentration for 148000 g/mol chitosan in 80% acetic acid in water. The entanglement concentration (ce) is 2.9 wt % and is determined by the change in slope (scaling exponent) on the above log-log plot. The inset shows how ηsp decreases after the solutions have been stored at room temperature for 1 month (red).

especially challenging because the amine group is protonated under acidic solutions. Therefore, chitosan is a cationic polyelectrolyte in acidic solutions, resulting in a much higher solution viscosity then a neutral polymer of similar size. Electrospun fibers form only when sufficient chain entanglements are present. However, as concentration is raised, viscosity increases and the solution may become too viscous to electrospin. To successfully electrospin a polymer, the concentration should be at least 2-2.5 times above the entanglement concentration (ce) so that a continuous polymer fiber can be formed.21,41 To determine ce for our chitosan sample, specific viscosity (ηsp ) (η0 - ηs)/ηs, where η0 is the zero shear rate viscosity and ηs is the solvent viscosity) data are plotted in Figure 2. Solutions span nearly two decades in concentration and a crossover from the semidilute unentangled to the semidilute entangled regime is observed as a change in slope. The ce was determined to be 2.9 wt %. In semidilute unentangled solution, this investigation found ηsp ∼ c1.3. This concentration dependence is in good agreement with the predicted relationship (ηsp ∼ c1.25).42 In semidilute entangled solution, this study found ηsp ∼ c6.0. This concentration dependence is much stronger than predicted (ηsp ∼ c3.75).42 This strong scaling dependence indicates the polymer chains are associating in solution.43,44 Cho et al.43 demonstrate that for chitosan with a degree of deacetylation of 93% and a molecular weight of 850000 g/mol, the ηsp scales with concentration to the 4.1 power for entangled systems. The discrepancy between their reported exponent of 4.1 and our observed 6.0 is most likely due to chitosan’s aging in solution (Figure 2 inset). If the chitosan solutions are aged at room temperature over a period of 1 month, then the slope of the concentration dependence of the viscosity, above ce, is decreased to 4.4 (ηsp ∼ c4.4, Figure 2 inset). Kampf et al.45 have also observed a similar reduction in viscosity over time for chitosan and describe the biopolymer as transforming from bundles of rigid rods to flexible polymer chains (based upon light scattering and molecular modeling results). Above ce, the rod bundles probably interact or associate with one another (based upon ηsp ∼ c6.0). As the bundles of rods transform into flexible coils, the chains are entangled but are no longer associating and, therefore, ηsp ∼ c4.4. Below ce, little change in ηsp is observed over time (Figure 2 inset). The hydrodynamic volume of the independent (nonassociating)

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bundles of rods may be similar to the hydrodynamic volume of the resulting flexible coils; if this is the case, there would be little change in ηsp below ce. Alternately, the chitosan chains may reach their equilibrium conformation more quickly below ce (i.e., the transition from bundles of rods to flexible coils occurs on the order of days as opposed to weeks). Additionally, if successful electrospinning only occurs at 2 to 2.5 times the ce, then the minimum necessary concentration of chitosan is from 5.8 to 7.25 wt %, with resulting viscosities ranging from 4000 to 16000 cP. Therefore, when attempting to electrospin chitosan solutions at concentrations above ce, the electric field is insufficient to overcome the combined effect of the viscosity and surface tension of the solution. In short, solutions above 2ce do not form a Taylor cone, while solutions that do form a Taylor cone (c < 2ce) produce beads rather than fibers (Figure 1). Beads form below ce, because there are no entanglements or topological constraints present and the jet from the Taylor cone cannot withstand the force of the electric field or surface tension of the solution and the jet breaks into droplets, which in turn form beads. In contrast to Geng et al.,24 we were unable to produce chitosan nanofibers from an acetic acid/water solvent system. The primary difference between this study and Geng’s is that the chitosan we used has a higher degree of deacetylation, about 80% versus 54%, which greatly impacts the viscosity of the resulting solutions. Geng et al. used chitosan with a molecular weight of 106000 g/mol and a degree of deacetylation of 54%, and they were able to electrospin uniform chitosan fibers.24 However, the viscosity of their chitosan solution at 5 wt % is about 600 cP, where the viscosity of the samples used in this study (molecular weight of 148000 g/mol and degree of deacetylation of 80%) at 5 wt % is 4530 cP, nearly an order of magnitude higher. Although the chitosan used by De Vrieze et al.35 is seemingly identical to what is used in this study, subtle differences in the degree of deacetylation can greatly alter its electrospinnability. Increasing the degree of deacetylation, increases the amount of free amine, which in turn increases the amount of charge on the chain under acidic conditions. As the charge on the chain increases the conformation of the chain in solution expands and the viscosity increases substantially. Therefore, the degree of deacetylation is an extremely important parameter to consider when attempting to electrospin chitosan. Chitosan/PEO Blended Solutions. To produce bead-free, chitosan-based nanofibers, chitosan was blended with poly(ethylene oxide) (PEO). The replacement of some of the chitosan with PEO (thereby, maintaining the same total polymer concentration) allows the solution as a whole to be more easily electrospun by offsetting chitosan’s high viscosity in the acetic acid/water solvent system.36,46 PEO has a low toxicity, is readily cleared by the body, has a minimal reaction in vivo and can be blended with chitosan in solution without decreasing the potential for biomedical end applications of an electrospun nanomesh.28 For the same overall polymer concentration, the addition of PEO reduces total solution viscosity, which creates a blend that is much more spinnable than chitosan alone while still resulting in a potentially biocompatible nanofibrous mesh. Early attempts at electrospinning PEO-chitosan blended solutions lead to the creation of blended fibers but were not free of defects (Figure 3a-d). Samples A-D (Figure 3 and Table 2) illustrate the subtle nature of this technique. Samples A and C differ only in acetic acid concentration in the final solution (A, 45%; C, 40%). The same is true for samples B (36%) and D (32%). The acetic acid concentration will alter both the conformations of the polymers in solution and the

Klossner et al.

Figure 3. Electrospun chitosan-PEO nanofibrous structures illustrating the effect of acetic acid concentration, chitosan-PEO ratio, and total polymer concentration: (a) 2:3 chitosan-PEO blend with 2.5 wt % total polymer and 45% total acetic acid; (b) 4:9 chitosan-PEO blend with 2.6 wt % total polymer and 36% total acetic acid; (c) 2:3 chitosan-PEO blend with 2.5 wt % total polymer and 40% total acetic acid; (d) 4:9 chitosan-PEO polymer ratio with 2.6 wt % total polymer and 32% total acetic acid; (e) 2:3 chitosan-PEO polymer ratio with 3.0 wt % total polymer and 32% total acetic acid; and (f) 8:9 chitosan-PEO polymer ratio with 3.4 wt % total polymer and 32% total acetic acid. Note: (a) is sample A in Tables 1 and 2; (b) is sample B, and so on. Magnification is 20000×.

conductivity of the solution, which in turn will affect how the solution electrospins and the morphology of the resulting structures (nanofibers). In this case, reducing the acetic acid concentration has a positive effect, reducing the number of bead defects in the electrospun fibers. By increasing polymer concentration and, thus, solution viscosity, the number of defects such as beads are reduced in many polymer-solvent systems.42,47 With that in mind, the total polymer concentration was increased from about 2.5-3 wt % (Figure 3e) and 3.4 wt % (Figure 3f), resulting in nearly defectfree nanofibers. The ratio of chitosan to PEO is identical (2:3) for samples A, C, and E. In contrast to samples A and C, sample E is essentially a defect-free nanofibrous web with an average fiber diameter of 129 ( 16 nm. The elimination of beads in sample E is due to the increase in overall polymer concentration and a further decrease in acetic acid concentration from 45% (A) to 40% (C) to 32% (E). Once defect-free, chitosan-containing nanofibers were produced (Figure 3e), the amount of chitosan in the nanofibers was increased. By increasing the chitosan to PEO ratio from 2:3 to 8:9 and increasing the overall polymer concentration from 3.0 to 3.4 wt % (refer to samples E and F in Table 2 and Figure 3), defect-free nanofibers with a diameter of only 62 ( 9 nm were formed. These were the smallest fibers produced in this study. In order to further increase the amount of chitosan in the nanofibers, the concentration of chitosan in the blended solutions was increased from 1.6 to 2.5 wt %. This change increased the total amount of polymer in the solution from 3.4 to 4.0 wt % (samples F-H, Table 2). Defect-free nanofibers of over 60% chitosan were produced, as illustrated in Figure 4.

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Table 2. Fiber Diameter and Solution Properties for Chitosan-PEO Blends sample A B C D E F G H a

CSa conc.

PEO conc.

1.0 0.8 1.0 0.8 1.2 1.6 2.0 2.5

1.5 1.8 1.5 1.8 1.8 1.8 1.8 1.5


% % % % % % % %


% % % % % % % %

polymer ratio CS/PEOb 2:3 4:9 2:3 4:9 2:3 8:9 10:9 5:3

resulting polymer conc. (CS + PEO)

AAa conc.

fiber diameter

2.5 2.6 2.5 2.6 3.0 3.4 3.8 4.0

45% 36% 40% 32% 32% 32% 32% 40%

178 ( 27 nm 168 ( 31 nm 172 ( 32 nm 121 ( 17 nm 129 ( 16 nm 62 ( 9 nm 112 ( 5 nm 103 ( 7 nm


% % % % % % % %

AA and CS represent acetic acid and chitosan, respectively. b The molecular weight of the chitosan and PEO was 148000 and 900000, respectively.

Figure 5. Sample F electrospun 1 day after blending (left) and the same solution electrospun 1 week after blending (right). Sample F has a 8:9 chitosan-PEO polymer ratio with 3.4 wt % total polymer and 32% total acetic acid. Figure 4. Nanofibers containing 62.5% chitosan with an average fiber diameter of 103 ( 7 nm (sample H: 5:3 chitosan-PEO blend with 4 wt % total polymer and 40% acetic acid).

Generally, as polymer concentration is increased the resulting fiber diameter also increases. Interestingly, that is not the case for this system. Excluding sample F, the fiber diameter of the remaining samples that formed bead-free nanofibers (E, G, and H) decreased as total polymer concentration (meaning the combined chitosan/PEO concentration) increased. If the fiber diameter is normalized by the percentage of chitosan in the nanofiber, the normalized fiber diameter increases with concentration. We hypothesize that this interesting behavior is due to the fact that chitosan is a polyelectrolyte and that the positively charged chain behaves differently in the electrical field than a neutral chain. This is the subject of continued investigation. Phase Separation of Blended Solutions. The blended solutions were able to be electrospun with the weakest electric field and the least amount of complications when the electrospinning was attempted within 24 h of initially being blended. Solutions that were electrospun one day after being blended required much less electric field strength to overcome the polymer droplet’s surface tension and viscosity than the same solution one week later (Figure 5). If the blended solutions were stored for more than 24 h, they (1) become increasingly difficult to electrospin, (2) require a higher electric field, (3) form beaded structures, and (4) eventually are no longer able to be electrospun. This behavior is due to phase separation, even though the solutions appear homogeneous to the eye for over 2 weeks. Viscosity measurements were used to probe the solutions’ behavior as a function of time. Four different blended solutions (Table 3) were tested as a function of time. The zero shear rate viscosity (η0) of these solutions decreases significantly after only several days (Figure 6), a factor which can be attributed to phase separation of the two component solutions. After 2 weeks to 1 month, solid polymer aggregates were visible upon close inspection when the solutions were swirled. Recalling the time-dependent behavior of chitosan in aqueous acetic acid solutions (Figure 2), we speculate that as the chitosan transforms from bundles

Table 3. Solution Properties of Samples for Time-Dependent Rheological Investigation ratio of CS solution resulting to PEO resulting polymer polymer conc. CSa conc. solutionb AAa conc.c ratio CS/PEO (CS + PEO) 3% 3% 5% 5%

50:50 60:40 50:50 60:40

40% 48% 40% 48%

1:1 1:1 5:3 5:3

3.0 3.0 4.0 4.2

wt wt wt wt

% % % %

a AA and CS represent acetic acid and chitosan, respectively. b All PEO solutions were 3 wt % in deionized water. The molecular weight of the chitosan and PEO was 148000 and 900000, respectively. c Acetic acid concentration in the chitosan component was 80%.

Figure 6. Zero shear rate viscosity (η0) of chitosan/PEO solutions (Table 3) measured as a function of time. The decrease in η0 indicates the solutions are phase separating over the course of several days.

of rigid rods to flexible polymer chains,45 it associates with the PEO in solution, eventually aggregating until it falls out of solution. Sodium chloride (NaCl) stabilizes the blended solutions, and solutions containing NaCl can be electrospun successfully for longer periods of time (Figure 7). Solutions that were spun

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Figure 7. PEO-chitosan 1:1 polymer solutions with 3.0 wt % total polymer and 40% acetic acid (one with 1.25 wt % NaCl (left) and one without (right)) were electrospun after being stored for 1 week at room temperature. NaCl stabilizes the solutions and allows them to be stored before use for a longer period of time.

successfully within 1 day of being blended would not form a Taylor cone (and therefore could not be electrospun) after 1 or more weeks of storage at room temperature. However, solutions that contained 1.25 wt % NaCl still were able to be electrospun after 1 week with limited defects (Figure 7). The addition of salt alters the confirmation of polyelectrolytes in solution by screening the charges covalently bound to the backbone of the chain. In the high salt limit (i.e., when salt ions greatly outnumber counterions), polyelectrolytes are in the same universality class as neutral polymers in good solvents and their concentration dependencies of viscosity, relaxation time, and terminal modulus are the same.42 Therefore, the addition of 1.25 wt % NaCl is altering the conformation of the chitosan chains in solution and, perhaps, influencing its transformation from bundles of rigid rods to flexible coils.45 The NaCl also may change the conformation of the PEO chains in solution, if the addition of salt changes the solvent quality (in this case, improving the solvent quality and stabilizing the PEO in the aqueous acetic acid). In any case, the addition of salt slows the aggregation and subsequent phase separation of chitosan and PEO in aqueous acetic acid.

Summary Electrospinning chitosan with high degrees of deacetylation (DD) is especially challenging because it is a cationic polyelectrolyte, resulting in a much higher solution viscosity then a neutral polymer of similar chain length. As the charge on the chain increases, the conformation of the chain in solution expands and the viscosity increases substantially. Therefore, the degree of deacetylation is an extremely important parameter to consider when attempting to electrospin chitosan. Indeed, when we attempted to electrospin chitosan with a DD of about 80% from aqueous acetic acid at concentrations above ce, the electric field was insufficient to overcome the combined effect of the viscosity and surface tension of the solution. In contrast, Geng et al.24 were able to electrospin chitosan from aqueous acetic acid, but their sample’s DD was only 54% and it’s viscosity was roughly an order of magnitude lower than our sample at the same concentration. Chitosan-PEO blends can be electrospun to create a mesh constructed of defect-free, nanofibers with diameters ranging from 62 to 129 nm, which would be suitable for use in biomedical applications. Of the samples studied, chitosan with the lowest molecular weight (148000 g/mol) was most suitable to electrospin. Increasing the total polymer (chitosan + PEO) concentration in solution reduces the number of beads, while increasing the chitosan concentration decreases fiber diameter. For our chitosan-PEO blends, reducing the acetic acid concentration from 45% to about 30% reduced the number of bead

Klossner et al.

defects in the electrospun fibers, possibly by altering the conformations of the polymers and the conductivity of the solutions. Over time, the chitosan-PEO blended solutions phase separate and are unable to be electrospun. As a result, blended solutions should be electrospun within 24 h of initially being blended to minimize complications. The addition of NaCl stabilizes the blended solutions and increased the time the blended solutions could be stored before electrospinning. Additional studies will focus on maximizing the amount of chitosan in the nanofibers and on the time dependence of both chitosan and blended chitosan-PEO solutions. Acknowledgment. The authors thank Dale Batchelor for his invaluable help with the SEM. We also thank the NCSU Nanotechnology Initiative and the Textile Engineering, Chemistry and Science Department at North Carolina State University, for providing funding for this research.

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