This work is funded in part by the USDA National Institute for Food and Agriculture, National Competitive Grants Program, National Research Initiative Program 71.1 FY 2007 as Grant No. 2007-35503-18392, and National Institutes of Health, Institute for Allergy and Infectious Disease, R33AI94514-03.

1. Spinning Dope Preparation
<ol>
	<li>Prepare a range of biopolymer concentrations to be investigated (0.1% to 30%, w/v) and be sure to consider moisture content of the biopolymer powder in these calculations. For each concentration, weigh the biopolymer (starch or pullulan) powder into a 50 ml test tube. Add aqueous dimethyl sulfoxide (DMSO) solution and a stir bar.</li>
	<li>Place the tube into boiling water with constant stirring on a magnetic stirrer hotplate.</li>
	<li>After about 1 hr, turn off heat and allow the dispersion to cool to room temperature. The dispersion is then ready for rheological testing and electrospinning.</li>
</ol>
2. Steady Shear Rheology
<ol>
	<li>Warm up the rheometer and set the stage temperature at 20 &#176;C. Calibrate the gap between the probe (25 mm cone used) and the stage (plate).</li>
	<li>Load 0.41 ml of the biopolymer dispersion onto the center of the stage and lower the probe to set the position (0.053 mm gap for 25 mm cone). Make sure that the dispersion evenly spreads within the gap.</li>
	<li>Perform the rheological test with the following experimental parameters: sweep mode: log, initial rate: 100 sec-1, final rate: 0.1 sec-1, points per decade: 10, delay before measure: 5 sec, measure time: 10 sec, and directions per measure: two (both clockwise and counterclockwise).</li>
	<li>Analyze rheological data to estimate appropriate dispersion concentrations for electrospinning trials.
	<ol>
		<li>Plot apparent shear viscosities against shear rates as a function of polymer concentrations. For each flow curve, approximate zero shear viscosities, &#951;0, by the actual or extrapolated values (e.g., at low concentrations where the low shear rate data are unreliable) for apparent viscosity at 0.1 sec&#8722;1.</li>
		<li>Calculate specific viscosity: &#951;sp = (&#951;0&#8722; &#951;s)/&#951;s, where &#951;s is the viscosity of the solvent.</li>
		<li>Plot specific viscosities as a function of concentration. Identify semidilute unentangled and entangled regimes. The semidilute unentangled regime begins from the low concentration end with a small slope, and the semidilute entangled regime has a greater slope following the unentangled regime. Fit power-law regression models in both regimes. The power values are the slopes (concentration dependence) in semidilute unentangled and entangled regimes on a log-log plot. The intersection of the two fitted lines is the entanglement concentration, ce.</li>
	</ol>
	</li>
</ol>
3. Electrospinning Parameter Variation
<ol>
	<li>Assemble the electrospinning setup as shown in Figure 1. Load the syringe with dispersion of appropriate composition, e.g., 15% (w/v) starch or pullulan in 100% DMSO, onto the syringe pump. Clip the high voltage wire (positive) to the needle. Connect the coagulation bath containing pure ethanol to ground by immersing the ground wire (negative) into the bath. Use a lab jack to adjust the distance between the syringe needle and coagulation bath. Immerse a metal mesh in the bath to collect the fiber mat after electrospinning.</li>
	<li>Spin the biopolymer in the following parameter ranges: feed rate from 0.1 to 0.4 ml/hr, spinning distance from 5 to 10 cm, and voltage from 0 to 15 kV.
	<ol>
		<li>Start with a spinning distance of 5 cm. For the first feed rate (0.1 ml/hr), ramp the voltage up slowly from 0 V. Pay attention to the shape of the dispersion extruded at the needle tip and note when the dripping dispersion is accelerated and then elongated.</li>
		<li>Note the voltage at which a tiny jet initiated from the drop surface, indicating electrospinnability of the solution. Record the voltage at which a continuous jet initiates, if any.</li>
		<li>Examine the complete range for each of the three parameters and note successful spinning conditions. Collect the fibers only when there is a continuous jet from the tip.</li>
	</ol>
	</li>
	<li>After a few minutes of collecting, rinse the fiber mat with pure ethanol. Place the fiber mat into a desiccator containing desiccant under vacuum.</li>
	<li>Repeat for each biopolymer concentration for complete characterization.</li>
</ol>
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51933/51933fig1highres.jpg" width="500" /> 
Figure 1.&#160;Schematic drawing of the electro-wet-spinning setup. The biopolymer dispersion is extruded from a syringe pump. A high voltage DC power supply provides high voltage to the blunt needle and grounds the coagulation bath. The polymer jet from the needle tip travels through a straight path and then develops a rapid whipping path (aka whipping instability).
4. Morphological Characterization
<ol>
	<li>Cut a piece of dried fiber mat and immobilize it onto a SEM stub using carbon tape.</li>
	<li>Load the sample stub into the SEM instrument and obtain images for analysis.</li>
</ol>

<ol>
	<li>Greiner, A., Wendorff, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+self-assembled+nanofibers+by+electrospinning.">Functional self-assembled nanofibers by electrospinning.</a> Self-aseembled nanomaterials. 1, 107-171 (2008).</li><li>Ramakrishna, S., Fujihara, K., Teo, W. E., Lim, T. C., Ma, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> An Introduction to Electrospinning and Nanofibers. , (2005).</li><li>Klossner, R. R., Queen, H. A., Coughlin, A. J., Krause, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+Chitosan&#8217;s+Rheological+Properties+and+Its+Ability+to+Electrospin.">Correlation of Chitosan&#8217;s Rheological Properties and Its Ability to Electrospin.</a> Biomacromolecules. 9 (10), 2947-2953 (2008).</li><li>McKee, M. G., Wilkes, G. L., Colby, R. H., Long, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlations+of+Solution+Rheology+with+Electrospun+Fiber+Formation+of+Linear+and+Branched+Polyesters.">Correlations of Solution Rheology with Electrospun Fiber Formation of Linear and Branched Polyesters.</a> Macromolecules. 37 (5), 1760-1767 (2004).</li><li>McKee, M. G., Hunley, M. T., Layman, J. M., Long, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solution+rheological+behavior+and+electrospinning+of+cationic+polyelectrolytes.">Solution rheological behavior and electrospinning of cationic polyelectrolytes.</a> Macromolecules. 39 (2), 575-583 (2006).</li><li>Kong, L., Ziegler, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+pure+starch+fibers+by+electrospinning.">Fabrication of pure starch fibers by electrospinning.</a> Food Hydrocolloids. 36, 20-25 (2014).</li><li>Singh, R. S., Saini, G. K., Kennedy, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pullulan:+microbial+sources,+production+and+applications.">Pullulan: microbial sources, production and applications.</a> Carbohydrate Polymers. 73 (4), 515-531 (2008).</li><li>Leathers, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biotechnological+production+and+applications+of+pullulan.">Biotechnological production and applications of pullulan.</a> Applied Microbiology and Biotechnology. 62 (5), 468-473 (2003).</li><li>Karim, M. R., Lee, H. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+characterization+of+electrospun+pullulan/montmorillonite+nanofiber+mats+in+aqueous+solution.">Preparation and characterization of electrospun pullulan/montmorillonite nanofiber mats in aqueous solution.</a> Carbohydrate Polymers. 78 (2), 336-342 (2009).</li><li>Stijnman, A. C., Bodnar, I., Hans Tromp, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning+of+food-grade+polysaccharides.">Electrospinning of food-grade polysaccharides.</a> Food Hydrocolloids. 25 (5), 1393-1398 (2011).</li><li>Kong, L., Ziegler, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+molecular+entanglements+in+starch+fiber+formation+by+electrospinning.">Role of molecular entanglements in starch fiber formation by electrospinning.</a> Biomacromolecules. 13 (8), 2247-2253 (2012).</li><li>Kong, L., Ziegler, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rheological+aspects+in+fabricating+pullulan+fibers+by+electro-wet-spinning.">Rheological aspects in fabricating pullulan fibers by electro-wet-spinning.</a> Food Hydrocolloids. 38, 220-226 (2014).</li><li>Han, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fiber Spinning Rheology and Processing of Polymeric Materials: Volume 2: Polymer Processing. , 257-304 (2007).</li><li>Morris, E. R., Cutler, A. N., Ross-Murphy, S. B., Rees, D. A., Price, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+and+shear+rate+dependence+of+viscosity+in+random+coil+polysaccharide+solutions.">Concentration and shear rate dependence of viscosity in random coil polysaccharide solutions.</a> Carbohydrate Polymers. 1 (1), 5-21 (1981).</li><li>Thompson, C. J., Chase, G. G., Yarin, A. L., Reneker, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+parameters+on+nanofiber+diameter+determined+from+electrospinning+model.">Effects of parameters on nanofiber diameter determined from electrospinning model.</a> Polymer. 48 (23), 6913-6922 (2007).</li></ol>

The authors declare that they have nothing to disclose.

Rheology is an essential tool to study the processing of polymers, including conventional fiber spinning and electrospinning13. From the steady shear rheological studies, polymer conformation and their interactions in different solvents can be resolved (Figures 2&#160;and&#160;3). At concentrations not high enough for biopolymer molecules to overlap with one another, their concentration dependence was around 1.4 (Figure 3), which was in good agreement with reported values of other polymers in good solvent3,4. After the biopolymer molecules start to entangle, the specific viscosity showed a much higher dependence on concentration. A greater n value indicates a stronger intermolecular interaction. Many random coil polysaccharides showed similar concentration dependence, with an n value of approximately 3.3&#160;14. Pullulan showed stronger interaction than starch in solvents of high DMSO content, possibly due to the molecular nature of the two biopolymers. The starch used had some highly branched components (~20% amylopectin), while the pullulan should be linear. Of course, the molecular weights, which were unknown, would also have an influence.
The entanglement concentration would depend on the conformation of the biopolymer in the dispersion. For example, the entanglement concentration of starch in 92.5% (w/v) DMSO aqueous solution is much lower than that in pure DMSO11. It implies that starch molecules exist in a more extended conformation in 92.5% (w/v) DMSO aqueous solution so that they occupy a larger hydrodynamic volume and tend to overlap more easily. The entanglement concentrations of pullulan did not vary as drastically as those of starch with varying solvent quality, probably because both water and DMSO are good solvents for pullulan and have little effect on the molecular conformation. Water, which is not a good solvent for starch, made the scenario much complicated, since undissolved starch molecules would affect the rheological response.
To spin good fibers, the concentration had to be 1.2-2.7 and 1.9-2.3x&#160;the entanglement concentration for starch and pullulan, respectively (Figures 4 &#38; 5). This range is narrower for pullulan, probably also due to less conformation difference in the solvents. It was interesting to note that a dispersion at entanglement concentration, when polymers start to entangle with one another, was not electrospinnable. Probably, high shear force involved in electrospinning impede chain overlap and long range polymer interaction that might have already been established at static and low shear conditions, and thus an enhanced and sufficient entanglement is required. In addition, shear viscosity also played an important role (Figure 6). The electrospinnable starch and pullulan dispersions fall into a similar range of shear viscosity at 100 sec-1, with an upper boundary of 2.2 Pa&#183;sec.
The procedure described herein can be modified in correspondence with the equipment and materials used in other studies. The dissolution of polymers is the first critical step in this protocol, because we found that partially dissolved starch dispersions (e.g., in 85% (w/v) DMSO) produced unstable steady shear viscosity data that prevented an accurate determination of ce. While conducting steady shear measurements, we prefer to start from the highest shear rate. By doing so, the dispersion is evenly distributed within the gap by the help of a high shear rate. The electrospinning step required much practice. Attention should be paid to the shape change of the droplet at the needle tip. Safety precautions during electrospinning should not be neglected. The main danger of electrospinning comes from the high voltage used in the process, although the current is relatively low. Electrospinning experiments should be performed in a fume hood in order to expel solvent vapor that may pose health hazards if one is exposed to it for a long time. Avoid close distance and even contact between the charged needle tip and the coagulation bath, because these will result in a short and fire hazard.
The rheological methods employed in the current study do have limitations. For instance, it should be noted that the actual shear rate involved in electrospinning is much higher than 100 sec-1&#160;1. In addition to shear rheology studied, elongational rheology, which characterize the stretching of dispersion along the trajectory, may also play an important role15. The rheometer used in this study is not capable of characterizing elongational viscosity.
Rheological studies can provide valuable information on biopolymer conformation in dispersions and their processing properties. This protocol is potentially useful in electrospinning of many other biopolymers and their blends, in terms of solvent system selection, optimization of parameters, and fiber forming mechanism on a molecular level.

Electrospinning is a technique that is capable of producing continuous micro- to nano-scale fibers from a wide variety of materials. It has gained increasing academic and industrial interest1. Though the setup and practice of electrospinning seem straightforward, the ability to predict electrospinnability and control fiber properties remains a challenge. The reason may lie in the fact that there are many factors influencing the electrospinning process2 and the process, especially the path travelled by the fiber, is chaotic1. Often an empirical &#8220;cook-and-look&#8221; approach is used for screening potential electrospinnable materials. However, to gain better control over the electrospinning process and resultant fiber properties, a more complete understanding of the mechanisms that govern electrospinnability is required. Several researchers have found that molecular entanglement of polymers in the spinning dope is an essential prerequisite for successful electrospinning3-5.
Rheology is a powerful tool to probe molecular conformation and interaction in polymer dispersions. For instance, McKee et al. investigated the molecular conformation of linear and branched poly(ethylene terephthalate-co-ethylene isophthalate) copolymers in a solvent containing chloroform/dimethyl terephthalate (7/3, v/v), and determined that the polymer concentration had to be 2-2.5x&#160;the entanglement concentration for successful electrospinning4.
There is currently renewed interest in fibers from biopolymers because of their advantages in biodegradability, biocompatibility, and renewability vis-&#224;-vis their synthetic counterparts. Yet practitioners confront many challenges arising generally from their structural complexity, difficulty in thermal processing and inferior mechanical properties. Starch, found in plant tissues, is among the most abundant and inexpensive biopolymers on earth. Pure starch fibers fabricated using an electro-wet-spinning apparatus were recently described6. Pullulan is a linear polysaccharide produced extracellularly by certain bacteria. The regular alternation of (1&#8594;4) and (1&#8594;6) glucosidic bonds are believed to be responsible for several distinctive properties of pullulan, including excellent fiber/film forming capability7,8. Electrospinning of pullulan fibers from aqueous dispersion has been reported by a number of researchers9,10. In our previous publications, the electrospinnability of two biopolymers, starch11 and pullulan12, has been discussed. This report focuses on demonstrating the protocol for utilizing rheological principles in the investigation of the electrospinnability of these two biopolymers.

<table><tbody><tr><td>Gelose 80 starch</td><td>Ingredion</td><td></td><td>Used as it is</td></tr><tr><td>Pullulan</td><td>Hayashibara Co. Ltd</td><td></td><td>Used as it is</td></tr><tr><td>Dimethyl sulfoxide</td><td>BDH Chemicals</td><td>BDH1115-4LP</td><td></td></tr><tr><td>Ethanol</td><td>VWR International</td><td>89125-172</td><td>200 proof</td></tr><tr><td>Rheometer</td><td>TA Instruments</td><td>ARES&amp;nbsp;</td><td>50 mm cone and plate geometry</td></tr><tr><td>Syringe (10 ml)</td><td>Becton, Dickinson and Company</td><td>309604</td><td>Syringe with Luer-Lok&amp;reg; Tip</td></tr><tr><td>High voltage generator</td><td>Gamma High Voltage Research, Inc.</td><td>ES40P</td><td></td></tr><tr><td>Syringe pump</td><td>Hamilton Company</td><td>81620</td><td></td></tr><tr><td>Environmental scanning electron microscope</td><td>FEI Company</td><td>Quanta 200</td><td>for starch fibers</td></tr><tr><td>Environmental scanning electron microscope</td><td>Phenom-World</td><td>Phenom G2 Pro</td><td>for pullulan fibers</td></tr></tbody></table>

molecular entanglement and electrospinnability of biopolymers

Electrospinning is a fascinating technique to fabricate micro- to nano-scale fibers from a wide variety of materials. For biopolymers, molecular entanglement of the constituent polymers in the spinning dope was found to be an essential prerequisite for successful electrospinning. Rheology is a powerful tool to probe the molecular conformation and interaction of biopolymers. In this report, we demonstrate the protocol for utilizing rheology to evaluate the electrospinnability of two biopolymers, starch and pullulan, from their dimethyl sulfoxide (DMSO)/water dispersions. Well-formed starch and pullulan fibers with average diameters in the submicron to micron range were obtained. Electrospinnability was evaluated by visual and microscopic observation of the fibers formed. By correlating the rheological properties of the dispersions to their electrospinnability, we demonstrate that molecular conformation, molecular entanglement, and shear viscosity all affect electrospinning. Rheology is not only useful in solvent system selection and process optimization, but also in understanding the mechanism of fiber formation on a molecular level.

Flow curves of the biopolymer dispersions as a function of biopolymer concentration and DMSO concentration in solvent were obtained. Two representative figures show the flow curves of starch (Figure 2A) and pullulan (Figure 2B) as a function of their concentration in pure DMSO solvent. The specific viscosities were plotted against biopolymer concentration (Figure 3A&#160;for starch and Figure 3B&#160;for pullulan). From these plots, entanglement concentrations were obtained as the intercept of the fitted lines in the semidilute unentangled and the semidilute entangled regimes.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51933/51933fig2highres.jpg" width="500" /> 
Figure 2.&#160;Flow curves of (A) Gelose 80 starch and (B) pullulan in pure DMSO as a function of concentration (%, w/v) at 20 &#176;C. In both figures, starch and pullulan of low concentrations were less viscous to produce sufficient torque at low shear rates. These unreliable data were thus not plotted. In general, the two biopolymers showed Newtonian behavior at low concentrations, i.e. the apparent shear viscosity was independent of shear rate. Shear thinning became apparent as their concentration increases, especially beyond 10% (w/v). Yet the shear thinning behavior was weak. The 15% and 20% (w/v) pullulan dispersions only showed the early phase of the power law region upon high shear rates, while the starch dispersions did not show significant reduction in viscosity over the shear rate range from 0.1 to 100 sec-1. Reprinted with permission from ref 11, Copyright (2012) American Chemical Society, and with permission from ref 12, Copyright (2014) Elsevier.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51933/51933fig3highres.jpg" width="500" /> 
Figure 3.&#160;Plot of specific viscosity versus (A) Gelose 80 starch and (B) pullulan concentration in pure DMSO. The slopes of the fitted lines in semidilute unentangled (left side) and the semidilute entangled (right side) regimes indicate the concentration dependence of specific viscosity, aka scaling dependence4. Pullulan showed stronger concentration dependence than starch in the entangled regime. The intercept of the two fitted lines was termed as the entanglement concentration (ce) at which biopolymers start to overlap in the dispersion. Starch required a higher concentration than pullulan to start entangling. Reprinted with permission from ref 11, Copyright (2012) American Chemical Society, and with permission from ref 12, Copyright (2014) Elsevier.
Electrospinning was attempted for all of the biopolymer dispersions, and results judged in terms of electrospinnability, i.e. jet forming ability during electrospinning, and morphology of the fibers formed. A dispersion of good electrospinnability formed a stable and continuous jet that resulted in continuous and smooth fibers without droplets. A dispersion that was not able to electrospin could not form a stable jet or develop whipping instability. Either tiny droplets or thick fibers were deposited into the coagulation bath. Figure 4 shows representative good and poor fibers evaluated from their appearance. Figure 5 summarizes evaluation of electrospinnability at varying concentrations of DMSO in solvent and the biopolymer in dispersion for starch and pullulan, respectively. In addition to entanglement concentrations, shear viscosities at 100 sec-1 were plotted against biopolymer concentration, where regions of electrospinnability were denoted (Figure 6).
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51933/51933fig4highres.jpg" width="500" /> 
Figure 4: Scanning electron micrographs of good (left) and poor (right) starch and pullulan fibers. Good fibers are smooth, continuous, and randomly oriented, while poor fibers may have beads, breaks, and droplets as shown in the figure (red circles). (a) 10% (w/v) Gelose 80 starch in 95% (v/v) DMSO, (b) 8% (w/v) Gelose 80 starch in 80% (v/v) DMSO, (c) 17% (w/v) pullulan in 40% (v/v) DMSO, and (d) 9% (w/v) pullulan in 80% (v/v) DMSO. Reprinted with permission from ref 11, Copyright (2012) American Chemical Society, and with permission from ref 12, Copyright (2014) Elsevier.
<img alt="Figure 5" fo:content-width="5in" src="/files/ftp_upload/51933/51933fig5highres.jpg" width="500" /> 
Figure 5.&#160;Evaluation of electrospinnability of (A) Gelose 80 starch and (B) pullulan dispersions as a function of DMSO concentration in solvent and biopolymer concentration in the dispersion: good electrospinnability (circles), poor electrospinnability (diamonds), and unable to electrospin (X&#39;s). Shaded areas roughly represent electrospinnable regions. Entanglement concentrations are also approximately labeled. Reprinted with permission from ref 11, Copyright (2012) American Chemical Society, and with permission from ref 12, Copyright (2014) Elsevier.
<img alt="Figure 6" fo:content-width="5in" src="/files/ftp_upload/51933/51933fig6highres.jpg" width="500" /> 
Figure 6.&#160;Shear viscosity (at 100 sec-1) of (A) Gelose 80 starch and (B) pullulan dispersions as a function of biopolymer concentration in different DMSO/water solvents. Shaded areas roughly represent the electrospinnable region. Reprinted with permission from ref 11, Copyright (2012) American Chemical Society, and with permission from ref 12, Copyright (2014) Elsevier.

Watch this Scientific Journal Video about Molecular Entanglement and Electrospinnability of Biopolymers at JoVE.com

Molecular Entanglement and Electrospinnability of Biopolymers

Electrospinning is a fascinating technique used to fabricate micro- to nano-scale fibers from a wide variety of materials. Molecular entanglement of the constituent polymers in the spinning dope is essential for successful electrospinning. We present a protocol for utilizing rheology to evaluate the electrospinnability of two biopolymers, starch and pullulan.

Electrospinning is a fascinating technique to fabricate micro- to nano-scale fibers from a wide variety of materials. For biopolymers, molecular ...

molecular-entanglement-and-electrospinnability-of-biopolymers

Nanyang Technological University

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JoVE Journal

Bioengineering

13.7K Views.  Pennsylvania State University. Electrospinning is a fascinating technique used to fabricate micro- to nano-scale fibers from a wide variety of materials. Molecular entanglement of the constituent polymers in the spinning dope is essential for successful electrospinning. We present a protocol for utilizing rheology to evaluate the electrospinnability of two biopolymers, starch and pullulan.

Video: Molecular Entanglement and Electrospinnability of Biopolymers

Electrospinning Fundamentals: Optimizing Solution and Apparatus Parameters

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. Electrospinning is a process in which a charged polymer jet is collected on a grounded collector; a rapidly rotating collector results in aligned nanofibers while stationary collectors result in randomly oriented fiber mats. The polymer jet is formed when an applied electrostatic charge overcomes the surface tension of the solution. There is a minimum concentration for a given polymer, termed the critical entanglement concentration, below which a stable jet cannot be achieved and no nanofibers will form - although nanoparticles may be achieved (electrospray). A stable jet has two domains, a streaming segment and a whipping segment. While the whipping jet is usually invisible to the naked eye, the streaming segment is often visible under appropriate lighting conditions. Observing the length, thickness, consistency and movement of the stream is useful to predict the alignment and morphology of the nanofibers being formed. A short, non-uniform, inconsistent, and/or oscillating stream is indicative of a variety of problems, including poor fiber alignment, beading, splattering, and curlicue or wavy patterns. The stream can be optimized by adjusting the composition of the solution and the configuration of the electrospinning apparatus, thus optimizing the alignment and morphology of the fibers being produced. In this protocol, we present a procedure for setting up a basic electrospinning apparatus, empirically approximating the critical entanglement concentration of a polymer solution and optimizing the electrospinning process. In addition, we discuss some common problems and troubleshooting techniques.

Electrospinning techniques can create a variety of nanofibrous scaffolds for tissue engineering or other applications. We describe here a procedure to optimize the parameters of the electrospinning solution and apparatus to obtain fibers with the desired morphology and alignment. Common problems and troubleshooting techniques are also presented.

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. ...

Air Filter Devices Including Nonwoven Meshes of Electrospun Recombinant Spider Silk Proteins

Based on the natural sequence of Araneus diadematus Fibroin 4 (ADF4), the recombinant spider silk protein eADF4(C16) has been engineered. This highly repetitive protein has a molecular weight of 48kDa and is soluble in different solvents (hexafluoroisopropanol (HFIP), formic acid and aqueous buffers). eADF4(C16) provides a high potential for various technical applications when processed into morphologies such as films, capsules, particles, hydrogels, coatings, fibers and nonwoven meshes. Due to their chemical stability and controlled morphology, the latter can be used to improve filter materials. In this protocol, we present a procedure to enhance the efficiency of different air filter devices, by deposition of nonwoven meshes of electrospun recombinant spider silk proteins. Electrospinning of eADF4(C16) dissolved in HFIP results in smooth fibers. Variation of the protein concentration (5-25% w/v) results in different fiber diameters (80-1,100 nm) and thus pore sizes of the nonwoven mesh.
Post-treatment of eADF4(C16) electrospun from HFIP is necessary since the protein displays a predominantly &alpha;-helical secondary structure in freshly spun fibers, and therefore the fibers are water soluble. Subsequent treatment with ethanol vapor induces formation of water resistant, stable &beta;-sheet structures, preserving the morphology of the silk fibers and meshes. Secondary structure analysis was performed using Fourier transform infrared spectroscopy (FTIR) and subsequent Fourier self-deconvolution (FSD).
The primary goal was to improve the filter efficiency of existing filter substrates by adding silk nonwoven layers on top. To evaluate the influence of electrospinning duration and thus nonwoven layer thickness on the filter efficiency, we performed air permeability tests in combination with particle deposition measurements. The experiments were carried out according to standard protocols.

Spider silk fibers display extraordinary mechanical properties. Engineered Araneus diadematus Fibroin 4 (eADF4) can be processed into nonwoven meshes using electrospinning. Here, the eADF4 nonwoven meshes are used to improve the performance of air filtering devices.

Based on the natural sequence of Araneus diadematus Fibroin 4 (ADF4), the recombinant spider silk protein eADF4(C16) has been engineered. This highly ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Fabricating Degradable Thermoresponsive Hydrogels on Multiple Length Scales via Reactive Extrusion, Microfluidics, Self-assembly, and Electrospinning

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), their ultimate clinical use has been hampered by the lack of biologically-relevant degradation observed for most smart materials. This is particularly true for temperature-responsive hydrogels, which are almost uniformly based on polymers that are functionally non-degradable (e.g., poly(N-isopropylacrylamide) (PNIPAM) or poly(oligoethylene glycol methacrylate) (POEGMA)). As such, to effectively translate the potential of thermoresponsive hydrogels to the challenges of remote-controlled or metabolism-regulated drug delivery, cell scaffolds with tunable cell-material interactions, theranostic materials with the potential for both imaging and drug delivery, and other such applications, a method is required to render the hydrogels (if not fully degradable) at least capable of renal clearance following the required lifetime of the material. To that end, this protocol describes the preparation of hydrolytically-degradable hydrazone-crosslinked hydrogels on multiple length scales based on the reaction between hydrazide and aldehyde-functionalized PNIPAM or POEGMA oligomers with molecular weights below the renal filtration limit. Specifically, methods to fabricate degradable thermoresponsive bulk hydrogels (using a double barrel syringe technique), hydrogel particles (on both the microscale through the use of a microfluidics platform facilitating simultaneous mixing and emulsification of the precursor polymers and the nanoscale through the use of a thermally-driven self-assembly and cross-linking method), and hydrogel nanofibers (using a reactive electrospinning strategy) are described. In each case, hydrogels with temperature-responsive properties similar to those achieved via conventional free radical cross-linking processes can be achieved, but the hydrazone cross-linked network can be degraded over time to re-form the oligomeric precursor polymers and enable clearance. As such, we anticipate these methods (which may be generically applied to any synthetic water-soluble polymer, not just smart materials) will enable easier translation of synthetic smart materials to clinical applications.

Protocols are described for the fabrication of degradable thermoresponsive hydrogels based on hydrazone cross-linking of polymeric oligomers on the bulk scale, microscale, and nanoscale, the latter for preparation of both gel nanoparticles and nanofibers.

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), ...

Methods for the Self-integration of Megamolecular Biopolymers on the Drying Air-LC Interface

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and macro-structures, as seen in the cases of moving water in vascular bundles and moisturizing water in skin layers. In this study, we developed a method for assessing the effect of aqueous liquid crystalline (LC) solutions composed of biopolymers on drying. As LC biopolymers have megamolecular weight, we chose to study polysaccharides, cytoskeletal proteins, and DNA. The observation of biopolymer solutions during drying under polarized light reveals milliscale self-integration starting from the unstable air-LC interface. The dynamics of the aqueous LC biopolymer solutions can be monitored by evaporating water from a one-side-open cell. By analyzing the images taken using cross-polarized light, it is possible to recognize the spatio-temporal changes in the orientational order parameter. This method can be useful for the characterization of not only artificial materials in various fields, but also natural living tissues. We believe that it will provide an evaluation method for soft materials in the biomedical and environmental fields.

A method for the drying-induced self-integration of megamolecular biopolymers on the air-liquid crystalline interface is provided here. This methodology will be useful not only for understanding the macroscopic potentials of biopolymers, but also as an evaluation method for soft materials in biomedical and environmental fields.

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Vapor Phase Deposition of Electroactive Poly(3,4-ethylenedioxythiophene) onto Electrospun Commodity Polymer Nanofibers

This study investigates the preparation of polyacrylonitrile (PAN) nanofibers through electrospinning to create highly porous and strong materials for applications in water purification, electrocatalysis, and biomedicine. The uniformly white PAN nanofiber mats were cut into 2 cm x 2 cm coupons to ensure consistency. After electrospinning, these nanofibers were coated with an electroactive polymer (EAP) using chemical vapor deposition, with iron (III) chloride (FeCl3) serving as an oxidant for polymerizing 3,4-ethylenedioxythiophene (EDOT) into poly(3,4-ethylenedioxythiophene) (PEDOT). The study examined the impact of different FeCl3 concentrations on PEDOT deposition on the PAN coupons. PEDOT deposition led to an increase in coupon weight. Scanning electron microscopy (SEM) revealed increases in the diameter of the nanofibers treated with increasing FeCl3 oxidant concentration, although higher FeCl3&#160;concentrations caused inter-fiber bridging, implying a concomitant decrease in inter-fiber spacing. Energy dispersive X-ray spectroscopy (EDS) was used to confirm the presence of Fe, Cl, and S in the nanofibers, with sulfur content rising with FeCl3&#160;concentration used, suggesting increased PEDOT deposition efficiency with increasing oxidant concentration. Mechanical testing showed that PEDOT-coated PAN fibers had improved tensile strength and toughness in the hydrated state compared to pure PAN nanofibers. These results highlight the crucial role of FeCl3&#160;concentration in influencing the morphology and properties of PAN-PEDOT composites, enhancing their suitability for applications such as water purification, tissue engineering, biosensing, catalysis, and energy storage.

This paper describes a simple technique for coating electrospun PAN nanofibers with electroactive polymers using vapor deposition. The effects of the oxidant FeCl&#8323; on the deposition of poly(3,4-ethylenedioxythiophene) coatings on PAN were investigated to understand and optimize polymer growth, fiber diameter, and overall mechanical strength of the nanofiber mats.

This study investigates the preparation of polyacrylonitrile (PAN) nanofibers through electrospinning to create highly porous and strong materials for ...

Chemistry

Controlling Electrospun Polymer Morphology for Tissue Engineering Demonstrated Using hepG2 Cell Line

Electrospinning affords researchers the opportunity to fabricate reproducible micro to nanoscale polymer fibers. The 3D fibrous architecture of electrospun polymers is regarded as a structural imitation of the extracellular matrix (ECM). Hence, electrospun fibers fabricated from biocompatible polymers have been widely investigated by tissue engineering researchers for their potential role as an artificial ECM for guiding tissue growth both in vitro and in vivo. All cells are acutely sensitive to their mechanical environment. This has been demonstrated by the discovery of multiple mechanotransduction pathways intrinsically linked to the cytoskeletal actin filaments. The cytoskeleton acts as a mechanical sensor that can direct the functionality and differentiation of the host cell depending on the stiffness and morphology of its substrate. Electrospun fibers can be tuned both in terms of fiber size and morphology to easily modulate the mechanical environment within a fibrous polymer scaffold. Here, methods for electrospinning polycaprolactone (PCL) for three distinct morphologies at two different fiber diameters are described. The morphological fiber categories consist of randomly oriented fibers, aligned fibers, and porous cryogenically spun fibers, with 1 &#181;m and 5 &#181;m diameters. The methods detailed within this study are proposed as a platform for investigating the effect of electrospun fiber architecture on tissue generation. Understanding these effects will allow researchers to optimize the mechanical properties of electrospun fibers and demonstrate the potential of this technology more thoroughly.

This method provides the means to test different polycaprolactone fiber morphologies and topographies for the purpose of tissue engineering. Small and large fibers are fabricated with random orientations, aligned orientations, and also porous cryogenically electrospun structures and used as platforms for cell culture.

Electrospinning affords researchers the opportunity to fabricate reproducible micro to nanoscale polymer fibers. The 3D fibrous architecture of ...

Procedure for Fabricating Biofunctional Nanofibers

Electrospinning is an effective processing method for preparing nanofibers decorated with functional groups. Nanofibers decorated with functional groups may be utilized to study material-biomarker interactions i.e. act as biosensors with potential as single molecule detectors. We have developed an effective approach for preparing functional polymers where the functionality has the capacity of specifically binding with a model protein. In our model system, the functional group is 2,4-dinitrophenyl (DNP) and the protein is anti-DNP IgE (Immunoglobulin E). The functional polymer, &alpha;,&omega;-bi[2,4-dinitrophenyl caproic][poly(ethylene oxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-PEO-CDNP), is prepared by anionic living polymerization. The difunctional initiator utilized in the polymerization was prepared by electron transfer reaction of &alpha;-methylstyrene and potassium (mirror) metal. The 2-methoxystyrene monomer was added first to the initiator, followed by the addition of the second monomer, ethylene oxide, and finally the living polymer was terminated by methanol. The &alpha;,&omega;-dihydroxyl polymer [HO-PEO-P2MS-PEO-OH] was reacted with N-2,4-DNP-&isin;-amino caproic acid, by DCC coupling, resulting in the formation of &alpha;,&omega;-bi[2,4-dinitrophenylcaproic][poly(ethyleneoxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-PEO-CDNP). The polymers were characterized by FT-IR, 1H NMR and Gel Permeation Chromatography (GPC). The molecular weight distributions of the polymers were narrow (1.1-1.2) and polymers with molecular weights greater than 50,000 was used in this study. The polymers were yellow powders and soluble in tetrahydrofuran. A water soluble CDNP-PEO-P2MS-PEO-CDNP/ DMEG (dimethoxyethylene glycol) complex binds and achieves steady state binding with solution IgE within a few seconds. Higher molecular weight (water insoluble i.e. around 50,000) CDNP-PEO-P2MS-PEO-CDNP polymers, containing 1% single wall carbon nanotubes (SWCNT) were processed into electroactive nanofibers (100 nm to 500 nm in diameter) on silicon substrate. Fluorescence spectroscopy shows that anti-DNP IgE interacts with the nanofibers by binding with the DNP functional groups decorating the fibers. These observations suggest that appropriately functionalized nanofibers hold promise for developing biomarker detection device.

An efficient approach for preparing nanofibers decorated with functional groups capable of specifically interacting with proteins is described. The approach first requires the preparation of a polymer functionalized with the appropriate functional group. The functional polymer is fabricated into nanofibers by electrospinning. The effectiveness of the binding of the nanofibers with a protein is studied by confocal microscopy.

Electrospinning is an effective processing method for preparing nanofibers decorated with functional groups. Nanofibers decorated with functional groups ...