Determining the applicability of electrospun nanofibers as
antibiotic delivery systems for the treatment of plant bacterial
infections
Varun Kulkarni & Erik Warnquist
1.0: Abstract
Bacterial infections in crop plants are currently treated in highly inefficient and expensive manners.
Current techniques involve completely coating the surface of infected plants with antibiotics that are
potentially harmful to the environment. This can result in dangerous runoff that is not only hazardous to
the environment but inefficient in effectively targeting bacteria. Our project utilizes the process of
electrospinning to combine a common plant antibiotic, ampicillin sodium salt, with a widely available
transport protein, bovine serum albumin (BSA), into one nanofiber. Particles of such nanofiber meshes
have shown promise in penetrating cell walls; by targeting and penetrating specific afflicted cells, the
treatment will greatly reduce the runoff into the environment as well as the overall cost of curing afflicted
plants. We developed an 8.7 wt.% control solution in distilled water, with the components of the solute
being 85 wt.% BSA and 15 wt.% polyethylene oxide (PEO). We also developed two solutions that
incorporated ampicillin sodium salt: one solution dividing the 85 wt.% BSA into 42.5 wt.% ampicillin
sodium salt and 42.5 wt.% BSA, and the other dividing the 8.7 wt.% into 33.3 wt.% ampicillin sodium
salt, 33.3 wt.% BSA, and 33.3 wt.% PEO. Each solution was spun at a distance of 15cm from the
grounded collector and with a voltage of 15kV. The solution was loaded into a bevel-tipped needle.
Examination under a scanning electron microscope revealed that the fibers had significant globular
formation, thus demonstrating that the solutions may not have been capable of legitimate matrix
construction. Agrobacterium tumefaciens, a common plant bacterium, was grown on solidified agar and
was subjected to samples of the different fibers. No bacterial death caused by the introduction of fibers
was noted. We believe that the electrospinning process compromised the bioactivity of the ampicillin
sodium salt and/or the ability of the BSA to act as a carrier protein, thus inhibiting effective treatment. For
future trials we look to electrospin standardized fibers with a proper matrix construction, and then
administer a prepared BSA-ampicillin sodium salt solution, perhaps through soaking the porous fibers.
2.0: Introduction
The concept of electrospinning has recently gained much attention as a potential method of
delivering bioactive agents, such as drugs that could be used to treat bacterial infections. Electrospinning
is a process in which a solution is placed inside a capillary tube and is exposed to an electric field. The
surface of the liquid becomes charged, and the power of the electric field is increased. The surface of the
solution, in the shape of a hemisphere, is slowly pulled away from the capillary, creating a cone-like
shape known as the Taylor cone. The power of the field is still increasing, and a point is reached where
the electric charge overcomes the surface tension of the solution. A charged jet of fluid is released from
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the Taylor cone, and lands on a grounded collector. As the fluid exits the cone, it lands on random places
on the collector these random landing zones originate from the fact that the fluid within the cone is
spinning around with different trajectories. This spinning, or “whipping,” process causes the solvent to
evaporate, and the charged polymer fibers are left behind. The fibers are highly stretched and reduced in
diameter, which results in a very high surface area to volume. The quality of the fibers is heavily
dependent on the characteristics of the solution, such as viscosity and molecular weight (Frenot &
Chronakis, 2003).
Incorporating antibiotics into nanofibers is useful because of this high ratio, as it provides a large
amount of drug coverage compared to the volume. However, adding a drug to a solution can considerably
change its viscosity, surface tension, and conductivity, which may be problematic as varying any of these
factors may restrict or impede the formation of fibers. According to Buschle-Diller et al., factors of the
spinning solution that should be considered are, among other things, its miscibility (the ability of liquids
to dissolve in any quantities or ratios), compatibility, and evaporation rate (2007). Miscibility is
particularly important when a bioactive agent is being incorporated into a spinning solution, as a higher
miscibility would result in a more homogenous solution and a greater chance of successfully forming
fibers. Along with taking such factors into account, the location of the drug within the nanofiber also
plays a crucial role in determining its release characteristics (Buschle-Diller et al., 2007).
Other techniques have been developed in order to bypass the complexities of creating a solution
with an antibiotic already incorporated within it. One such technique is the plasma treatment method. This
technique involves the treatment of nanofibers with plasma to increase their surface adhesion. Plasma
treatments with air or argon have been used in particular because of their ability to be dissolved or wetted
by water. It is more convenient because it does not require producing a solution that retains the
antibacterial properties of a drug while being able to form nanofibers. Once the nanofibers are formed,
plasma treatment can be used to incorporate them with anti-bacterial properties. (Hyuk, Taek, & Park,
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2009). The antibacterial properties of the fibers from this treatment are very effective: 99.999% of
Staphylococcus aureus was killed after 4 hours of contact (Yao, Li, Neoh, Shi, & Kang, 2008).
However, even with these techniques, a solute for the solution is still required. One proposed
solute is albumin. Albumin is an abundant plasma protein that can be used as a solute to produce
nanofibers. Using albumin has many advantages; its stability in a range of pH levels, it’s resistance to
heat without damaging effects, and it’s biodegradability make it a strong candidate as a possible solvent.
Also, it is neither toxic nor immunogenic, which is beneficial (Kratz, 2008).
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Figure 1.0 Surface modification techniques for nanofibers (Hyuk, Taek, & Park, 2009)
It is also important to examine the different aspects of bacteria, specifically A. tumefaciens, in
order to determine the most effective method of combating them. Bacteria are prokaryotes, or
microscopic single celled organisms that are found in every habitat on earth. They are only micrometers
in length and come in a variety of shapes, including bacilli and cocci. The most adaptable and testable
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bacterium for laboratory purposes is the Agrobacterium tumefaciens. The A.tumefaciens is a pathogenic
bacterium that negatively affects the plant it inhabits. When infecting the plant it causes the disease
known as the crown gall, a very economically significant disease because the effects it has upon plants
calls for expensive treatments or removal that can further hamper the grower. The gall begins to develop
because of the way the bacterium inserts its DNA into the ribosome of the plant, thus causing the
overproduction of cytokinins, auxins, and opines. The auxins and cytokinins are responsible for the
growth of the gall tumor and the opines serve as nutrients for the bacteria. After infecting the plant,
cankerous tumors that deform the stem of the plant begin to appear. At the same time the metabolism of
the plant starts to become impaired due to the combination of the DNA of from the A.tumefaciens and the
infected plant (Collins, 2001). Specifically, the bacterium inserts a portion of its plasmid, which results in
uncontrolled cell division and eventually in the formation of the crown gall typical of A. tumefaciens
infections. Research also indicates that Agrobacterium can suppress the response of the plant immune
system to develop a more efficient system of transformation and infection (Ditt, Nester, & Comai, 2005).
A.tumefaciens is known to affect over 60 plant families throughout the world. The bacterium
belongs to the Rhizobiacae family and is classified as Gram-positive. Furthermore the structure of the
bacteria is the rod shaped and they grow aerobically within the plant without developing endospores.
When the A.tumefaciens are applied to a culture containing carbohydrate substance with large amounts of
extracellular polysaccharides the cultures will develop in fairly voluminous amounts with a slimy
appearance according to laboratory observations (Collins, 2001).
The bacteria A. tumefaciens can severely impair the plant it infects. However, this is not always
the case. Some bacteria are in fact essential for the development of some plants because of the way they
use nitrogen. Many bacteria are capable of extracting the nitrogen from the atmosphere and combining it
with hydrogen from the soil to create ammonium, which plays a vital role in the balance of the soil and
the ecosystem. Bacteria are an essential factor for the balance of nature, so it is essential to point out that
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preventive antimicrobial treatments should only be used on bacteria that negatively impact their host plant
(Knee & Nameth, 2007).
Antibiotic use on plants contributes to approximately 0.1% of the total antibiotic use in the United
States, reports Patricia McManus of the University of Wisconsin (2007). The majority of this amount is
dedicated to fruit producing crops such as pears, apples, and peaches. Other food plants common to the
usage of antibiotics are tomatoes, potatoes, peppers, and nectarines. Despite the recognition during the
1950’s and 1960’s that antibiotics could prove to be the miracle drug to cure all plant ailments, the
movement has encountered issues. The sheer price of developing the quantities needed to treat whole
acres has significantly outweighed the success antibiotics have been able to display throughout the years.
Because of the pricing issues associated with the use of antibiotics, growers in many regions depend on
whether based prediction systems that pin point the most efficient time to make use of the treatments.
Secondly, due to the emergence of resistances to the drugs, problems have occurred in the treatment of
plant bacterial infections. There are currently only two drugs registered by the EPA for safe usage on
plants, Streptomycin and Oxytetracycline. In recent years groups have rallied around the idea that through
the use of antibiotics on plants, the ability of life saving drugs to do their job in the future is significantly
decreasing. Growers whose lives depend on the livelihood of their crops counter the argument with the
idea that with the minimal amount of antibiotics used, no effect will take place (McManus, 2007).
A. tumefaciens can be easily cultured through the use of LB broth. This broth can be used in
either a solid induction method or a liquid induction method. One of the ideal strains of A. tumefaciens to
culture is AGL-1. However, it should be noted that this strain cannot grow with the assistance of different
antibiotics blended with the agar, which help repel other bacteria and overall contamination of the colony;
there may be a slightly higher risk of sample contamination in growing AGL-1 (Flowers & Vaillancourt,
2005).
The goal of this investigation was to develop a drug delivery system that incorporates an
antibiotic (ampicillin sodium salt) and a carrier protein (bovine serum albumin) within a nanofiber. By
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using nanofibers to administer drugs, the amount of antibiotics used would be significantly decreased.
Furthermore, using nanofibers as a drug delivery system is advantageous as it can target specific infected
locations rather than rather than rely on crude blanketing techniques. The bovine serum albumin, which is
a hydrophilic molecule with a hydrophobic cleft, was ideal as a transport molecule not only because of its
molecular structure but because of its hardiness; the molecule can withstand a pH range of 4-9 as well as
temperatures up to 60 degrees Celsius without any significant change in structure. It was hypothesized
that by blending the two components in a solution, the electrospinning process will have fused them
together into one fiber without compromising bioactivity. Several solutions were prepared, with the solute
components being BSA, ampicillin sodium salt, and polyethylene oxide (PEO), a common spinning
agent, and the solvent being distilled water. The subsequent fibers were examined under a scanning
electron microscope to observe fiber quality and fiber consistency. Also, samples of each fiber were taken
and applied to an in-vitro culture of A. tumefaciens to determine their bioactivity. A series of fibers
without an antibiotic component were used as a control to compare to the results of any experimental
fibers.
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3.0: Methodology
3.1: Solution Development
A control solution was developed, composing of bovine serum albumin (BSA), polyethylene
oxide (PEO), and distilled water (dH2O). The solution was composed with an 8.7 wt. % solute, which was
further broken down into 85 wt. % BSA and 15 wt. % PEO. The solvent was dH2O. Two alternative
solutions were also created, with each one incorporating ampicillin sodium salt, a common antibiotic for
plant bacterial infections. The first solution divided the 8.7 wt. % solute into 42.5 wt. % BSA, 42.5 wt. %
ampicillin, and 15 wt. % PEO. The second solution divided the solute into 33.3 wt. % BSA, 33.3 wt. %
ampicillin, and 33.3 wt. % PEO. Bovine serum albumin was obtained from Sigma Aldrich, product
number A7906. Ampicillin sodium salt was obtained from Sigma Aldrich, product number A0166. Both
were refrigerated at a constant temperature of approximately 4 degrees Celsius. PEO was obtained from
AOS.
3.2: Electrospinning Setup
Each of these solutions was exposed to identical electrospinning parameters. Every solution was
loaded into a 5mL syringe; the syringe was placed in a syringe pump. The needle used was a bevel-tipped
needle, and the flow rate was set at a constant 0.2mL/hr. The distance between the tip of the needle and
the grounded collector plate was 15cm. The solution was spun at 15 kilovolts. The collector plate was
made out of copper and was covered with aluminum foil; the foil provided a plane on which the fibers
could be collected.
Half of the electrospinning trials were spun with Flouroglide® sprayed onto the aluminum foil.
Flouroglide® is a Teflon like substance that eases the removal process when collecting the fiber for
bacterial trials. The Flouroglide® was sprayed onto the foil then dried via the use of a heat lamp for
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approximately 5 minutes. This allowed the spray to dry and thus not mix with the BSA solutions when
electrospinning.
3.3: Scanning Electron Microscopy
The resultant fibers were examined under a scanning electron microscope, which was located in
Janelia Farms research center (due to scheduling conflicts, the fiber from the solute composed of 42.5 wt.
% BSA, 42.5 wt. % ampicillin, and 15 wt. % PEO could not be examined). The fibers were examined
then two pictures were taken of each fiber respectively. Each picture was conducted at different
magnifications and was taken as to be representative of the fiber structure on the whole. Magnification
levels, pixel size, and relevant measurements of structures were recorded into appropriate laboratory
notebooks.
3.4: Bacterial Preparation
Solid agar plates were prepared using a dry agar solution from Scholar Chemistry. The agar was
combined with water in the ratio of 23.3 grams of agar to one liter of dH2O. Large stock supplies of agar
were prepared, autoclaved to remove unwanted bacterial growth, then stored at 8 Celsius in the
refrigerator for later use. The cooled agar solution was then heated on a hot plate. A sterile work
environment was prepared through the use of 10% bleach on lab surfaces. Then a Bunsen burner was lit
to circulate air and prevent the landing of new bacteria onto the agar. The solution was then poured into
the Petri dish, while keeping the top of the Petri dish at a 45 angle for further bacterial growth protection.
The agar was allowed to re-solidify, usually via a 10-25 minute waiting period. Once the agar was solid, a
solution consisting of Luria Broth (LB) and dH2O was prepared in the ratio of 35 grams of solid LB agar
base to one liter of dH2O. A 100 ml solution was prepared using 3.5 grams of the LB agar base.
Agrobacterium tumefaciens from Carolina® was then removed from refrigeration. An inoculating loop
removed from sterile packaging was the swabbed across the surface of the A. tumefaciens nutrient agar
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and swirled around in the LB solution. The other non-used end of the inoculating loop was then used to
remove more bacteria and was also swirled around in the LB solution. A sterile L-shaped spreader was
then immersed in the LB solution now impregnated with the A. tumefaciens. The spreader was then
removed from the solution and spread across the surface of the agar, making sure to cover the entire
surface. The Petri dish was subsequently closed then sealed with Parafilm® for the control sample.
For the trials using the electrospun nanofibers the bacteria was applied to the Petri dishes in the
same fashion. Prior to sealing however, these plates were treated with four respective treatments.
Treatments were prepared by first pealing the fibers from the aluminum foil through the careful use of
tweezers and scalpels. Once removed the fibers were cut into relatively similarly sized two dimensional
pieces. Each piece was approximately a 0.5 cm by 0.5 cm square. On each plating four treatments were
placed in a square formation (see below).
Figure 3.4: Example of plating method
with fibers located on the surface
For each trial two control fiber treatments of the same batch were used on the plate for two of the spots.
For the other two spots two treatments of the two trial concentrations were used (of the same batch). Four
trials of two dishes each were used all together.
3.5: Data Recognition
To examine the results for bacterial death, the process of region of inhibition was chosen. In this
method the spread of bacterial death around the treatment it measured. The treatments were placed far
enough apart in the hopes that the regions would not intersect thus further disturbing the data. The regions
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were created, their respective diameters were measured. These diameters were compared to those of the
control and between trial groups.
4.0: Results
4.1: Fiber Images
Spun on: February 15th, 2010
Solution: 8.7 wt% control fiber: 85 wt. % BSA, 15 wt. % PEO
Magnification: 2.61 x 103
Sample #: 1
Figure 4.1.1
Figure 4.1.1 is the first in a series of scanning electron images of the fibers that were developed. The
solution that the fiber was created from, an 8.7 wt. % solute with 85 wt. % BSA and 15 wt. % PEO, was a
control as it does not incorporate ampicillin sodium salt. The image shows significant globular formation,
with clusters of spherical globs heavily concentrated around several narrow, thin strands of fiber. The thin
strands are characteristic of the fibers generated from a pure PEO solution, so it was determined that the
globular formations were composed of electrospun BSA. The overall lack of the development of a
consistent BSA fiber is not favorable, in that inconsistent globs are indicators of a structurally unstable
mesh. However, in this context of drug delivery, the globular formations may be beneficial, as they may
have the potential to carry the ampicillin molecules within the fiber.
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Spun on: February 15th, 2010
Solution: 8.7 wt% control fiber: 85% BSA, 15% PEO
Magnification: 1.15 x 103
Sample #: 1
Figure 4.1.2
Figure 4.1.2 shows a different sample of a fiber
of the same composition as Figure 4.1.1. The
conclusions drawn about the fiber from Figure
4.1.1 are supported in this image. The globs of
BSA still seem to follow the thin strand of PEO
fibers, although not with enough consistency to
form a clear nanofibrous mesh. This sample also
lacked any continuous fiber composed of BSA.
Spun on: February 15th, 2010
Solution: 8.7 wt% control fiber: 85% BSA, 15% PEO
Magnification: 1.19 x 103
Sample #: 2
Figure 4.1.3
Figure 4.1.3 is another image of a sample of the
same control solution. This image shows a
clearly defined clustering of the globular protein
formations around specific patches of the PEO
mesh. However, there is still no continuous and
consistent fiber.
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Spun on: February 15th, 2010
Solution: 8.7 wt% control fiber: 85% BSA, 15% PEO
Magnification: 8.18 x 103
Sample #: 2
Figure 4.1.4
Figure 4.1.4 is a magnification of a patch of the
previous sample. It is clear to see that the
locations of each glob of BSA are somewhat
influenced by the position of the PEO meshes,
but that influence is not enough to completely
create a fiber with a PEO core and BSA globs as
an external layer.
Spun on: February 15th, 2010
Solution: 8.7 wt% control fiber: 85% BSA, 15% PEO
Magnification: 3.63 x 103
Sample #: 2
Figure 4.1.5
The figure on the previous page is another
image of the same sample. Again, the trends
outlined in the previous images are
consistently being supported in this image.
The globular formations seem to share a
collinear pattern similar to that of the PEO
mesh that is closest to them, but the
correlation is not strong enough to develop a
neat fiber.
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Spun on: March 9th, 2010
Solution: 8.7 wt% control fiber: 85% BSA, 15% PEO
Magnification: 1.21 x 103
Sample #: 3
Figure 4.1.6
As seen with the previous control fibers, very
little fibrous formation was evident in this
updated sample. Nearly no connection matrices
are visible between the globs of BSA. They
seem to be conglomerating around the “fibers”
of PEO. However, even with the assistance of
the spinning agent, very little fibrous
construction can be observed in this sample.
Spun on: March 9th, 2010
Solution: 8.7 wt% control fiber: 85% BSA, 15% PEO
Magnification: 8.83 x 103
Sample #: 3
Figure 4.1.7
The large globular structures appear to be BSA,
as PEO is a highly stable and distinct fiber when
spun. The average size of the globs appears to be
approximately 897.1 nm. Accompanying the
globs are shredded forms of the PEO.
Essentially, this shows no distinct fiber
formation; the image seen is a clustering of the
solution prepared into the electrospinning
machine.
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Spun on: March 9th, 2010
Solution: 8.7 wt% test fiber: 33.3% BSA, 33.3% PEO, and 33.3% ampicilin
Magnification: 1.56 x 103
Sample #: 4
Figure 4.1.8
In this image, it seems that the globs seem to be congregating more heavily around the portions of PEO.
This could be due to the increase of the concentration of PEO within the solute. This is logical, as an
equal wt. % of BSA and PEO within the solution would most likely yield a more consistent fiber, even if
the fiber is not regular. Another important observation is the smeared effect of the globs of the BSA. It is
possible that the addition of the ampicillin into the mix somehow morphed what was occurring in the
BSA and stretched the globs, although this may also be a side effect of balancing the wt. % of the BSA
and the PEO. The fibers in this image also seem to have protruding shards of what may be ampicillin (the
shape is not similar to either spun BSA or PEO). The difference in the texture of the fibers, as compared
to the previous images, may denote a coating of the fibers with the morphed BSA. It is important to note
that the globs seem to be located in significant clusters, which are highlighted by the circles; the
clustering is similar to the correlation between PEO concentration and BSA conglomeration seen in the
previous images. Overall, the addition of ampicillin in the solution as well as the balance between the
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BSA concentration and the PEO concentration have yielded favorable results; this image shows more
distinct fibers that are potentially lined with ampicillin and coated with BSA. Optimally, such a fiber may
receive protection from the BSA coating and remain bioactive from the ampicillin shards.
Spun on: March 9th, 2010
Solution: 8.7 wt% test fiber: 33.3% BSA, 33.3% PEO, and 33.3% ampicilin
Magnification: 5.50 x 103
Sample #: 4
Figure 4.1.9
At first glance this image has a striking resemblance to portion B of figure 1.0 in which the surface graft
polymerization process is described. This image is promising because at a more magnified view, 5.50 x
103 magnification, it can be seen that there is indeed some fiber formation. Although the bioactivity of
such structures cannot be confirmed, it does appear that there is a PEO base fiber with globs of
BSA/ampicillin connected in branching formations. This is further supported by the measurement of the
glob, 935.7 nanometers in length, which is highly similar to the sizing of the BSA glob measured in
sample 3 (Figure 4.1.7.).
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4.2: Bacterial Results and Analysis
Figure 4.2.1
The pictures above(figure 4.2.1) is an images of a colony of Agrobacterium tumefaciens being
treated with a shard of a fiber that was created from a solute of 15 wt. % PEO, 42.5 wt. % BSA, and 42.5
wt. % ampicillin (not shown in the image above). The rationale for such a solution is that the resultant
fiber will contain equal concentrations of BSA and ampicillin, in such a way that the amount of antibiotic
in the solution is maximized while preserving the 15 wt. % PEO as a spinning agent.
The treatment revealed some mixed results. The colony is obviously affected by the treatment, as
the texture and the color of the bacteria definitely changed (the colony on the right was left untreated as a
control; the color of the treated and the untreated bacteria clearly differs). The shard that was used as a
treatment also seemed to be directly consumed by the bacteria, as it is completely coated with bacteria.
No bacterial death was observed, and no zones formed. The growth of the bacteria over the treatment
actually indicates that the nanofiber was ineffective in killing/preventing bacterial growth. In other words,
this evidence goes against the hypothesized bioactivity of the fiber. The color change may or may not be a
sign of bioactivity, as color change can result from a variety of causes, such as a fungal contamination. As
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there was no way to confirm whether or not the color change is a result of the bioactivity of the fiber, the
test was inconclusive.
The trend observed here is representative of all the other data. In fact, to put the images of the
plates covered with thriving bacteria would be useless. The results found on the agar trials were so
decisive that they did not even warrant a picture. Neither concentration of fibers provided any bacterial
death. This would demonstrate an error in process of methodology that will be discussed later in the
discussion section (5.0). Furthermore even the positive control we conducted provided no results. We
created a base control solution of 8.7 wt% 85% BSA and 15% PEO then immersed sterile filter paper
circles of 0.5 mm radius into the solution. We prepared an agar via the L-shaped spreader then placed the
circles onto the bacteria in the same formation we did with the fiber treatments. After placement into the
incubator and 2 day waiting period, once again there was absolutely no bacterial death. This demonstrates
problems with the overall solution preparation if not others.
5.0: Discussion
The scanning electron microscope images of the different fibers were obtained from the Janelia
Farms Research Center. The fibers that were examined were all spun at a constant 15 kilovolt charge and
at a distance of 15 centimeters between the tip of the syringe and the grounded collector. The
concentrations and contents of the solutions were modified for different experimental fibers. Control
fibers were created from the control solutions with a solute of 8.7 wt %, which was further broken down
into 85 wt. % bovine serum albumin and 15 wt. % polyethylene oxide. The solvent was distilled water.
Trial fibers were composed from two trial solutions, with the solute remaining a constant 8.7 wt %: one
was 42.5 wt. % BSA, 42.5 wt. % ampicilin sodium salt, and 15 wt. % PEO, and the other was 33.3 wt. %
BSA, 33.3 wt. % ampicillin sodium salt, and 33.3 wt. % PEO. These two trial solutions were spun at the
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same voltage and distance from the grounded collector as the control. These fibers were collected on
aluminum foil and then examined via the SEM.
The images of the fibers can be seen in Figures 4.1.1 through 4.1.9. Analysis for each individual
fiber is provided beneath the images. However, when looking at the general fiber construction,
overarching conclusions can be drawn. Consistent throughout the solutions, there is very little fibrous
construction. Instead, globs of BSA with with relatively minute PEO connection fibers are apparent. This
may have been due to a lack of sufficient PEO in the solution as a polymerizing/fiber constructing agent,
the high viscosity of the solution, or other experimental/ambience flaws, such as humidity, stirring
techniques, etc. In any case, the fibers did not exhibit the characteristics of an optimum and stable fiber,
such as consistency and lack of beading. Despite this problem of legitimate fiber construction, the fibers
did have the potential to carry antibiotics. Figure 4.1.8 and figure 4.1.9 provide the best insight into the
formation of the fibers. Figure 4.1.8 shows how the addition of more PEO and ampicillin sodium salt into
the solution affected the fiber by stretching out the globs of BSA into more of a smear. It is believed that
this may have occurred primarily due to the addition of ampicillin, as PEO is known to form only narrow,
long fibers. Such an effect is unlikely to affect the BSA globs. Furthermore, PEO is not likely to interfere
with the spinning of any other component of the solution, as it is such a stable spinning agent. The
stretched out globs may be a result of modifying the solution with the addition of ampicillin sodium salt.
Figure 4.1.8 also shows significant clustering of globs, as indicated by the circles. This again may have
occurred because of the ampicillin or because of the balance in concentration between the BSA and PEO.
By having them in equal amounts, the PEO would have been more likely to create connections between
the globs and perhaps bring them into contact. Figure 4.1.9 gives a magnified image of the same sample.
This figure shows a construction very similar to that of portion B of Figure 1.0 (the surface graft
treatment). The similarity indicates that the image may indeed show a successfully treated fiber (albeit
through a blend solution method rather than through surface graft treatment). The differences in texture,
along with a comparison with the other control images, led to the conclusion that the fiber in the image
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may be a PEO base fiber that incorporates shards of ampicillin sodium salt and is coated by the BSA.
Such a combination is favorable because of the stability of the base and the hardiness of the coating. It
should be noted, however, that such images do not indicate the status of the fibers’ bioactivity. This is a
promising find and shows that for later studies, it may be of use to keep the polymer base and the other
components in equivalent ratios.
In order to test the efficacy of the treatments Petri dishes were plated with A. tumefaciens. The
dishes were plated via the lawn method it which the bacteria was introduced into a Luria broth solution.
This solution was then spread across the surface of the agar. This was done so that the bacteria would
grow consistently across the plate. Directly after being plated, 1 cm by 1 cm square portions of the fibers
were applied onto the plate as shown in figure 3.4. After this was done the plates were placed in the
incubator at 25 degrees Celsius. After remaining in the incubator for 48 hours the plates were removed.
Similar to image 4.2.1 treatments had absolutely no ability to kill bacteria. Despite some changes to the
color of the bacteria, the bacteria lived on successfully. This trend continued with all the trials of both
concentrations. Of the 10 agar dishes that were prepared, no trials demonstrated any sort of bacterial
death.
We proposed several hypotheses to describe why the fiber was unsuccessful in killing and
preventing the growth of A. tumefaciens. It was first hypothesized that the ampicillin sodium salt had lost
its bioactivity in the electrospinning process; exposure to 15kV may have altered the structure of the
antibiotic, thus rendering it ineffective. Another hypothesis stated that the colony of A. tumefaciens,
which has a rapid growth rate, may have a grown over the antibiotic after it had taken effect. This was a
possibility, as the effects of the antibiotic were not observed until 2 days after its application (a regular
drug-laden fiber will release approximately 99% of its drug in 4 hours). A third hypothesis stated that the
problem was with the original solution; the ampicillin may have lost its bioactivity before the
electrospinning process, or may have reacted unfavorably with the BSA or PEO. We also considered the
notion that the strain of bacteria that was being used may be an ampicillin-resistant strain. Several
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different tests were conducted to isolate the error to one of these variables. To eliminate the possibility of
the A. tumefaciens re-growing over our treatment, snapshots were taken 4 hours after the application as
well as a day after the application. Neither observation showed any initial zone of inhibition or re-growth.
As a result, we were able to eliminate the notion that the growth rate exceeded the effects of the
antibiotic. To determine whether the loss in bioactivity occurred during the electrospinning process, a trial
of positive controls was established. The idea of this trial was to see if the ampicillin sodium salt could
successfully kill and/or form a zone of inhibition in the culture before it was exposed to the high voltages
associated with electrospinning. To run the positive control, several cultures of A. tumefaciens were first
prepared, along with the control solution and the solution that had a balanced solute. We soaked separate
sets of filter paper in both solutions, and placed them in different locations in the different cultures.
Neither the control nor the experimental solution showed any bioactivity or effectiveness in handling the
bacteria. This demonstrates that the bioactivity may have been compromised even before the
electrospinning process, which indicates that either the bacterium is ampicillin-resistant or the solution is
interacting unfavorably. The source of error between these two options has not yet been confirmed. It is
also important to note that this testing did not rule out the possibility of denaturing through the
electrospinning process.
In conclusion, nanofibers still hold promise in integrating ampicillin sodium salt and bovine
serum albumin into a feasible drug delivery system. Based on the experimental scanning electron
microscope images, a basic fiber matrix can still be developed, with BSA and ampicillin as additions to
the base structure formed by the PEO. However, the basic matrix outline was only seen when the
components of the solute had a balanced concentration, which indicates that the solute must not be
dominated by one substance if it entails multiple agents. Also, the structural stability and the effectiveness
of the ampicillin sodium salt after being subject to the electrospinning process has not been examined; it
is possible that the extreme voltages may have affected the ability of the antibiotic to kill/prevent the
growth of A. tumefaciens.
21
For the future we plan to pursue to the ability of BSA to assist nano-scale drug delivery systems.
The best method for improvement would be to modify how the fibers are prepared in order to facilitate
the bacterial killing process. This can take on two pathways: either to focus on the biocompatibility of the
solution prior to spinning or to develop a structurally stable fiber first and then treat it to add the
antibiotic. Such a method is advantageous in that the sensitive antibiotic is able to bypass the high voltage
of the electrospinning process. If the solution can be seen to exhibit antibacterial properties prior to
spinning, then techniques such as surface graft polymerization will certainly hold promise in creating a
stable and bioactive fiber. Research also indicates that fibers, due to the nature of which they are created,
are responsive to electromagnetic fields. It may be possible to increase the targeting abilities of the
nanofibers by manipulating them with such fields, thus increasing their overall efficiency.
22
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