Electrospinning of Aligned PLGA Fibers

Alexander P. Cook
Introduction
Electrospinning is a common technique to manufacture nanofibers from a wide range of polymer
solutions. The advantages of electrospinning compared to other fiber fabrication techniques include
extremely long fiber length, controllable diameter (from the micro- down to nanometer range), large
surface area, porous structure, and molecular alignment.[1] There has been much research into biological
applications of electrospun fibers, such as drug delivery, biosensors, and tissue engineering.[2] In
particular, neural tissue engineering research has focused on understanding how neurons react to various
physical, electrical, and chemical guidance cues. Although much has been discovered, the effect of
nanorough surfaces on neuron growth remains to be elucidated.[3]
In this respect, aligned electrospun fibers are useful for creating nano-scale channels in which to guide
axon growth. The similarity of electrospun fibers to natural extracellular matrix (ECM) makes them ideal
substrates on which to study nerve regeneration.[3] In particular, poly(lactic-co-glycolic acid) (PLGA)
nanofibers are useful for this application due to their biocompatibility and biodegradability.[4]
The goal of this research was to create aligned PLGA nanofibers using standard electrospinning
techniques. Although aligned fibers have been achieved before in the literature[5], there are no papers (to
my knowledge) that have focused on PLGA as the polymer of interest. These PLGA fibers can then be
coated in poly(pyrrole) (PPy) to create the conductive substrates on which to plate neurons. Achieving
aligned PLGA fibers is the first step toward understanding the role that nanorough surfaces play in
directing neuron growth.
Materials and Methods
Solution Preparation
Polymer solutions of PLGA were prepared consisting of 14 weight % 85:15 PLGA and 2 weight %
BTEAC (salt) in chloroform (CHCl3). This solution was loaded into a 3 mL syringe with a 23-gauge
needle. These solution parameters were obtained from previous work by Ning.
Electrospinning
A standard horizontal electrospinning setup was used. An automated syringe pump dispensed the polymer
solution. The needle was connected to a high-voltage lead, and a mechanical spinning wheel was
electrically grounded. The substrates consisted of plastic microscope slides cut up and sputter coated with
100 nm of gold in the shape of the electrode. The substrates were attached to the wheel with double-sided
tape and grounded with aluminum foil. Humidity and temperature of the electrospinning environment
were noted.
Electrospinning was conducted under several variations of experimental parameters to determine their
effect on the alignment of the resulting PLGA fibers. Parameters tested included syringe flow rate,
applied voltage, wheel spin rate, and time. Based on the individual results, several combinations of these
parameters were tested together. Table 1 gives the details of each experiment. The control conditions
(also established from previous work by Ning) were 350 L/hr flow rate, 10 kV applied voltage, 1400
RPM wheel rate, 10 cm needle to substrate distance, and 10 min. of electrospinning time.

Table 1: A list of the values of variables used for the PLGA fiber alignment experiments.

Variable Tested
Syringe flow rate
Applied voltage
Wheel spin rate
Applied voltage at a lower flow rate

Values Tested
50, 150, 250, 350 L/hr
10, 12, 14, 16 kV DC
800, 1000, 1200, 1400 RPM
250 L/hr & 10, 12, 14, 16 kV DC

Inspired from work by Miriam, the method of electrically grounding the substrates to the wheel was also
tested. In a set of experiments, three conditions were investigated: a standard aluminum foil connection
from the gold directly to the wheel, no aluminum foil (the samples were not grounded in any way), and
aluminum foil taped directly above and below the substrate (but not touching the gold).
Additionally, a novel experiment was conducted based on an idea found in an electrospinning review
paper by Xia and Li.[1] They found that electrospinning poly(vinyl pyrrolidone) (PVP) fibers onto two
conductors separated by an insulating gap resulted in fiber alignment between the two conductive areas. I
decided to apply this idea to aligned PLGA fibers. For this experiment, a vertical electrospinning setup
was used. Two electrodes were grounded and separated by a distance of about 1 cm. Electrospinning was
conducted under the standard control parameters for several minutes. The plastic gap between the two
gold electrodes was examined to determine if fiber alignment had occurred between the two conductive
areas.
Microscopy
Samples were analyzed under an optical microscope to qualitatively determine the degree of PLGA fiber
alignment and density, as well as any other noteworthy characteristics. Pictures were taken at
magnifications of 5x, 20x, and 50x for each sample.
Results
Figures 1-3 show the results of varying the syringe flow rate. 50 L/hr did not yield significant fiber
density on the substrate and is not included.

Figure 1: 350 l/hr (20x)

Figure 2: 250 l/hr (20x)

Figure 3: 150 l/hr (20x)

Figures 4-7 show the results of varying the applied voltage.

Figure 4: 10 kV (20x)

Figure 5: 12 kV (20x)

Figure 6: 14 kV (20x)

Figure 7: 16 kV (20x)

Figures 8-11 show the results of varying the wheel spin rate.

Figure 8: 1400 RPM (20x)

Figure 9: 1200 RPM (20x)

Figure 10: 1000 RPM (20x)

Figure 11: 800 RPM (20x)

Figures 12-14 demonstrate the effect of the different grounding methods on fiber alignment.

Figure 12: Al foil ground (5x)

Figure 13: No ground (20x)

Figure 14: Al foil above and below ground (20x)

Finally, Figure 15 shows the result of using the vertical electrospinning setup with two substrates
separated by a small gap.

Figure 15: Two electrodes separated by a small insulating (plastic) gap (20x)

Discussion
When the flow rate was varied, the lower values yielded thinner fibers. 50 L/hr resulted in fibers so thin
that they were almost invisible to the naked eye during the electrospinning process. However, an
insignificant amount of fibers collected on the substrates in this case. Therefore, 50 L/hr is too slow of a
flow rate to achieve aligned PLGA fibers. Under the microscope, there was a directly proportional
relationship between the flow rate and the density of electrospun fibers (lower flow rates resulted in less
dense mats of fibers). The best alignment of PLGA fibers occurred for 150 L/hr. However, the lower
density of fibers at this value compared to 250 and 350 L/hr may skew the results (a higher density of
fibers means a greater chance for misalignment just on a statistical basis).
Based on observations during electrospinning, higher applied voltages resulted in finer fibers that were
ejected toward the substrates at higher velocities. There was also less clogging of the nozzle by the
viscous polymer solution at higher voltages. This is potentially due to the larger electrostatic force better
overcoming the surface tension of the liquid. Under the microscope, higher voltages gave higher fiber

densities. However, these higher voltages also tended to give larger numbers of misaligned fibers. There
was also a distinction noted in the nature of the misaligned fibers at different voltages. At 12 and 14 kV,
misaligned fibers tended to occur in patchy squiggles, whereas at 16 kV, misaligned fibers tended to be
more wavy and occur more in the direction perpendicular to the velocity of the spinning wheel. It is
unclear whether this was a random fluke or evidence of slightly better alignment at 16 kV. However,
overall there is no clear trend in alignment as a function of the applied voltage.
When the velocity of the spinning wheel was varied, there was generally a consistent density of fibers for
each trial. The alignment may be slightly better in the 1000-1200 RPM range. In general, 1400 RPM may
be a bit fast and 800 RPM a bit slow to achieve aligned fibers. Overall, no significant improvement in
PLGA fiber alignment was seen for these experiments.
When the lower flow rate was coupled with changing the voltage, similar results were seen compared to
the original voltage trials. Fiber density generally increased with higher voltages. The proportion of
aligned fibers did not improve in any significant way during these experiments compared to the voltage
trials at the original flow rate.
After talking to Miriam, I learned that she did not electrically ground her samples when electrospinning. I
decided to experiment with the method of grounding the gold to the wheel after having this discussion
with her. As seen in Figures 12-14, the method of grounding has a very significant effect on the outcome
of the aligned fibers. The regular aluminum foil ground I had been using up to that point results in lots of
squiggly, misaligned fibers (Figure 12). Without a ground, the alignment is actually very good (Figure
13). However, the density of fibers is not as good, probably because the gold surface itself lacks electrical
grounding. By placing aluminum foil above and below the substrate, this slightly increased the density of
fibers on the gold, but also introduced a bit more misalignment than without a ground entirely.
These results show that the main obstacle in achieving aligned PLGA fibers lies in grounding the
substrates. It appears that it is best to not ground with aluminum foil at all. The more aluminum foil that is
included in the setup (and especially if it is in contact with the gold), the greater the proportion of
misaligned fibers.
Figure 15 shows the result of using a vertical setup with two substrates separated by a short distance. The
alignment of PLGA fibers is fairly good. This is significant because the method mentioned by Xia and Li
in the review paper[1] has (as far as I know) not been attempted with PLGA fibers. This method is
advantageous because the wheel is no longer required to achieve aligned fibers. However, it is also
problematic because the aligned fibers must somehow be removed from the insulating gap and placed on
a fresh gold substrate. Xia and Li have written a paper[6] devoted entirely to this method of aligned fibers
that may be helpful if this topic should be pursued further. My experiments demonstrate that this
procedure could reasonably be used to achieve aligned PLGA fibers.
Overall, from the work I have conducted and what I have heard from Miriam, the best parameters for
achieving aligned fibers are 150 or 250 L/hr flow rate, 10 kV applied voltage, 1000 RPM wheel spin
rate, 10 cm needle to substrate distance, 23 gauge needle, and 10 min. electrospinning time. Humidity was
generally around 30-32 % and is unlikely to have had a significant effect on these results. Likewise,
temperature varied from around 25-28C depending on the day, but there is no evidence to believe that
this had a significant effect on the fiber alignment results.
Electrospinning is a difficult process to control. The combination of many variables combined with little
daily differences (e.g. temperature, humidity) at times made it difficult to pinpoint the factors responsible
for the observed alignment effects. By far, the most dramatic improvement was seen when the aluminum

foil grounding method was abandoned. Although there may have been some effects from experimental
variables (e.g. flow rate, voltage, spin rate, etc.), it is clear that the experimental setup is much more
crucial to achieve aligned PLGA fibers. Future work should focus on constructing a simple yet
sophisticated electrospinning setup that makes it easier to consistently execute trials. Only after the
equipment is set up sufficiently will it become truly possible to observe the effects of varying flow rate
and other experimental variables with confidence.
References
1. Li, Dan, and Younan Xia. "Electrospinning of nanofibers: reinventing the wheel?." Advanced
materials 16.14 (2004): 1151-1170.
2. Bhardwaj, Nandana, and Subhas C. Kundu. "Electrospinning: a fascinating fiber fabrication
technique." Biotechnology advances 28.3 (2010): 325-347.
3. Hoffman-Kim, Diane, Jennifer A. Mitchel, and Ravi V. Bellamkonda. "Topography, cell
response, and nerve regeneration." Annual review of biomedical engineering 12 (2010): 203.
4. Anderson, James M., and Matthew S. Shive. "Biodegradation and biocompatibility of PLA and
PLGA microspheres." Advanced drug delivery reviews 64 (2012): 72-82.
5. Yang, F., et al. "Electrospinning of nano/micro scale poly (L-lactic acid) aligned fibers and their
potential in neural tissue engineering." Biomaterials 26.15 (2005): 2603-2610.
6. Li, Dan, Yuliang Wang, and Younan Xia. "Electrospinning nanofibers as uniaxially aligned
arrays and layerbylayer stacked films." Advanced Materials16.4 (2004): 361-366.