Enhancing antibacterial
efficacy using protein
nanoparticles
Combined Research Paper
Leslie Tan Zheng Yu (4S1)
Tan Jing Chong (4S2)
Erik Warnquist (AOS)
Varun Kulkarni (AOS)
Hwa Chong Institution (High School)
Academy of Science, Virginia USA
Mentors: Ms Jennifer Ting Jeum Shian
Mr Jason Beltz
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 also inefficient in
effectively targeting bacteria. This study investigates the use of protein nanoparticles and
nanofibers as drug carriers to increase antibacterial efficacy of antibiotics against
Agrobacterium tumefaciens, the causal agent of crown gall disease that leads to the
formation of tumors in plants. Advantages of using protein nanoparticles and nanofibers as
drug carriers include an increased therapeutic efficacy, with controlled drug release and
increased localization through targeting and penetrating specific afflicted cells that will
greatly reduce the runoff into the environment.
Bovine Serum Albumin (BSA) nanoparticles are synthesized by simple coacervation
technique and the drug loading technique of antibiotics is varied. BSA nanofibers are
synthesized by electrospinning, with the concentration of BSA used varied. The comparison
of the zone of inhibition and number of bacterial colonies formed would be used to
investigate the antibacterial efficacy of the different drug delivery techniques.
No bacterial death caused by the introduction of fibers was noted, and it was
probably the electrospinning process that 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 nanoparticles, result showed that antibacterial efficacy of drug loaded by
encapsulation was the lowest. On the other hand, antibacterial efficacy of drug loaded by
surface adsorption was on par with pure antibiotics in the initial period of treatment. However,
the antibacterial efficacy decreased with increasing time of treatment. Surface adsorption
was the most effective developed technique for drug delivery.
2
Introduction:
Nanotechnology is making rapid progress in the development of materials with
suitable physical and chemical properties that makes it a potential tool in the medical field.
Nanotechnology has a plethora of useful applications and one such application would be
using nanoparticles as drug delivery systems. According to Allen et al., (2004), nanoparticles
can help improve the therapeutic properties of drugs by altering the biodistribution and
pharmacokinetics of the drug which will minimize the possible side effects. Drugs may cause
damage to healthy cells due to poor biodistribution but this can be solved through controlled
drug release which reduces the spread of the drug that reduces the effects on non-target
cells. Also, some drugs may be cleared from the body too rapidly and this makes it
necessary to increase drug dosages. With nanoparticles as drug carriers, drug clearance
can be reduced by altering the pharmacokinetics of the drug. Cells will take up drug loaded
nanoparticles due to its size, allowing the drugs through the cell membrane into the cell
cytoplasm. This increases the antibacterial property of the drug. Many diseases occur due to
processes within cells and they can only be stopped by drugs that enter the cell. In addition,
drugs with poor solubility can be carried by nanoparticles like liposomes with both hydrophilic
and hydrophobic environments.
Nanoparticles, such as liposomes, allows for controlled drug delivery. Liposomes can
encapsulate drugs within its lipid bilayer and adhere to the cell membrane of the target site
for drug delivery (Sobha et al., 2010). Studies by Rahimnejad et al., (2006) showed that
utilizing liposome as a drug carrier rather than giving regular doses of drugs reduce its
toxicity to healthy cells and minimize potential side effects. Thus, nanoparticles could be
incorporated into chemotherapy to abate any adverse effects. Also, nanoparticles release
drugs slowly, reducing the frequency of required treatment. At the same time, a reduction in
dosage allows for lower resistance of bacterial against antibiotics.
3
Nevertheless, liposome does have its disadvantages too. Liposomes have low
encapsulation efficiency. It was observed by Rahimnejad et al., (2006) that liposome also
has poor storage stability due to rapid leakage of water soluble drugs. An alternative to
liposomes as a drug delivery vehicle would be using Bovine Serum Albumin (BSA). Just like
viruses and plasmids, protein nanoparticles are promising as a drug carrier (Mostafa et al.,
2009). Both liposome and BSA can be prepared with ease. They improve the therapeutic
effects and reduce the side effects of the formulated drugs due to its controlled release
properties (Mohsen J and Zahra B 2008). However, BSA nanoparticles are sustainable and
well controlled for targeted drug delivery. Antibiotics do not leak as easily too. Other
alternatives include metallic nanoparticles such as silver nanoparticles. However, there is a
lack of research to show that such particles do not accumulate in the body which may result
in side effects. On the other hand, research by Rahimnejad et al., (2009) showed that BSA
nanoparticles are non-toxic, non-antigenic and biodegradable so it does not accumulate
indefinitely in tissues.
Given the potential of BSA nanoparticles as drug carriers, our research aims to
incorporate antibiotics into BSA nanodroplets and nanofibres for use in plants infected with
A.tumefaciens. While there are numerous research carried out over the application of
nanoparticles for chemotherapy, there is a lack of such research in plants. Nanofibres would
be formed through the electrospinning method. Nanodroplets would be formed through the
coacervation method. For the electrospinning method, the concentration of BSA would be
varied to find out the optimum condition for nanofibre formation. As for coacervation method,
the optimal drug loading technique for enhancing antibacterial efficacy of drug loaded
nanoparticles on A.tumefaciens is found. Both of them would then be subjected to in-vitro
testing. The nanodroplets and nanofibres would then be compared to see which one has a
higher antibacterial efficacy.
4
Such BSA nanoparticles would bring about both financial and environmental benefits
if they prove to be efficient against A.tumefaciens in plants. Conventional methods of dealing
with bacterial include crop dusting, which involves spraying antibiotics from an aircraft. This
could have a serious implication on the environment. Off-target contamination by antibiotics
due to spray drift and runoff from soil may result in damage to human health as well as land
and water contamination. Another concern is that the bacterial may gain resistance against
the antibiotics due to overuse. Not all of the antibiotics will target the target species, leading
to wastage of antibiotics and financial loss. However, with BSA nanoparticles, such
environmental damage and financial loss would be minimized simply because there is a
targeted delivery of antibiotics. Also, this minimizes the amount of antibiotics used, which will
minimize the possibility of resistance against the antibiotics.
5
Objective:
Create a more effective method for the treatment of plant bacterial through utilizing a carrier
protein (Bovine Serum Albumin) that can penetrate cell walls.
Hypothesis:
1. If nanoparticles and nanofibers both utilizing antibiotics are used to treat A.
tumefaciens, then one will prove to be more effective than the other.
2. Drug delivery by nanoparticles and nanofibers will affect the antibacterial efficacy of
the antibiotic.
3. Drug loading technique will affect the antibacterial efficacy of nanoparticles.
6
Methodology:
Materials Used:
Bovine Serum Albumin, Absolute Ethanol, 25% Glutaraldehyde, Polyethylene Oxide,
Deionised Water, A.tumefaciens, Kanamycin, Ampicillin, LB Broth/Agar, 0.20 μm cellulose
acetate membrane, Aluminum Foil, 10 ml Syringe.
Apparatus Used:
Centrifugal tubes, Beakers, Measuring Cylinders, Petri Dishes, Glass Stir Rods, 50
ml Graduated Cylinders.
Equipments Used:
3cm by 3cm Collector Plate, Power Source (up to 30 kV), Electrospinning Chamber,
Ring Stand, Scanning Electron Microscope, Hot Plate, Scale, Incubator, Centrifuge,
Micropipette, Magnetic Stirrer, Pipette Controller, Laminar Hood.
7
i.
Preparation of drug loaded BSA Nanoparticles
Coacervation technique was used for the preparation of BSA nanoparticles. Absolute
Ethanol was added to 15 ml of 1 % Bovine Serum Albumin solution at pH 7.0 until turbidity
was observed. 30 μl of 25% Glutaraldehyde was added and allowed to stir for 4 hours. The
mixture was centrifuged at 9,500 RPM for 30 mins and the supernatant was collected. It was
microfiltered through a 0.20 μm Cellulose Acetate Membrane to collect the BSA
nanoparticles.
Drug Loading by Encapsulation
Prior to addition of Absolute Ethanol, BSA solution was incubated with antibiotics at
concentration of 2% for one hour. Preparation of nanoparticles continued as mentioned
above.
Drug Loading by Surface Adsorption
BSA nanoparticles prepared as mentioned above were incubated with antibiotic
solution at concentration of 2% for one hour. Mixture was then centrifuged at 9,500 rpm for
30 mins and microfiltered through a 0.20 μm Cellulose Acetate Membrane to collect the drug
loaded nanoparticles.
ii.
Preparation of drug loaded BSA Nanofibers
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.
8
The second solution divided the solute into 33.3 wt. % BSA, 33.3 wt. % ampicillin, and 33.3
wt. % PEO.
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 approximately 5 minutes. This allowed the spray to dry and
thus not mix with the BSA solutions when electrospinning.
Scanning Electron Microscopy
The resultant fibers were examined under a scanning electron microscope. And 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.
iii.
Testing efficacy of drug loaded BSA nanoparticles
Zone of Inhibition
LB agar plates were first prepared. LB broth containing overnight culture of A.
tumefaciens was streaked on the surface of the LB plates through the use of a cotton swab.
Wells were punched on the agar and 50 μl of each sample were placed into respective well.
9
LB agar plates were incubated at 30oC for 48 hours and the diameter of the zone of
inhibition around wells after treatment were measured and recorded.
For negative control, BSA solution was replaced by 10mM Tris/HCl Buffer in the
formation of nanoparticles to show that leftover chemical reagents do not exhibit antibacterial
properties. Likewise, antibiotic was replaced by deionised water to show that BSA
nanoparticles on its own do not exhibit antibacterial properties.
Bleach, a known antibacterial chemical, was used as a positive control.
Pure antibiotic was used as another sample for comparison.
For each trial, samples included negative controls, positive control, pure antibiotics
and drug loaded nanoparticles by surface adsorption and encapsulation.
Total Plate Count
1ml of each sample was added to 8ml of LB broth and 1ml of A.tumefaciens. This
was repeated to obtain a total of 3 sets of each sample to be used in the test that was
conducted over a period of 2 days (Day 0, Day 1, Day 2). Samples were incubated at 30
degree Celsius. On their respective days, serial dilution was conducted before 100μl of each
sample was spread on LB agar plates. LB agar plates were incubated at 30oC for 48 hours
and the number of colonies formed were counted and recorded.
Control consisting of 1ml A.tumefaciens placed in 9ml LB broth for each day was
used for comparison with samples after treatment.
Pure antibiotic was also used as a sample for comparison.
For each trial, samples include control, pure antibiotics and drug loaded
nanoparticles by surface adsorption and encapsulation.
iv.
Testing efficacy of drug loaded BSA nanofibers
LB agar plates were first prepared. LB Broth containing culture of A.tumefaciens was
spread on the surface of the LB agar using an L-shaped spreader. Electrospun nanofibers
collected were cut into approximately 0.5 cm by 0.5 cm squares. Each piece was placed on
10
Figure 1: Example of plating
method with fibers located on
the surface
the LB agar as shown in Figure 1. LB agar plates were incubated at 30oC for 48 hours and
the zone of inhibition around wells after treatment were measured and recorded.
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.
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Results:
SDS-PAGE and SEM Images of Nanoparticles:
Figure 2: Comparison of SDS-PAGE results
Figure 3.1: SEM image of ampicillin loaded nanoparticles by surface adsorption at 10,000 X
magnification
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Bovine Serum
Albumin / Ampicillin
Figure 3.2: SEM image of ampicillin loaded nanoparticles by surface adsorption at 30,000 X
magnification
The SDS-PAGE result at the top shown in Figure 2 is obtained from research papers.
Comparing this with one of the SDS-PAGE result obtained shown at the bottom,
resemblances were observed, such as the protein bands being more refined and straight at
the sides.
Figure 3.1 shows clumping of nanoparticles, which correspond to previous
researches that concluded that clumping of particles occurs during the formation of
nanoparticles. Hence the particles shown in Figure 3.2 are believed to be ampicillin loaded
BSA nanoparticles.
13
SEM Images of Nanofibers:
Figure 4.1: SEM Image of 8.7 wt% control fiber: 85 wt. % BSA, 15 wt. % PEO at
magnification of 2.61 x 103 X
Figure 4.1 shows the SEM image of a control fiber without incorporating 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
fibers.
14
Figure 4.2: SEM Image of 8.7 wt% control fiber: 85% BSA, 15% PEO at magnification of
1.15 x 103 X
Figure 4.2 shows a different sample of a fiber of the same composition as Figure 4.1.
The conclusions drawn about the fiber from Figure 4.1 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.
15
Figure 4.3: SEM Image of 8.7 wt% control fiber: 85% BSA, 15% PEO at magnification of
1.19 x 103 X
Figure 4.3 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.
Figure 4.4: SEM Image of 8.7 wt% control fiber: 85% BSA, 15% PEO at magnification of
8.18 x 103 X
Figure 4.4 is a magnification of a patch of the sample shown in Figure 4.3. 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.
16
Figure 4.5: SEM Image of 8.7 wt% control fiber: 85% BSA, 15% PEO at magnification of
3.63 x 103 X
Figure 4.5 is another image of the same sample shown in Figure 4.3. Again, the
trends outlined in the previous SEM 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.
Figure 4.6: SEM Image of 8.7 wt% control fiber: 85% BSA, 15% PEO at magnification of
1.21 x 103 X
As seen with the previous control fibers, very little fibrous formation was evident in
this sample shown in Figure 4.6. Nearly no connection matrices are visible between the
globs of BSA. They seem to be conglomerating around the “fibers” of PEO. However, even
17
with the assistance of the spinning agent, very little fibrous construction can be observed in
this sample.
Figure 4.7: SEM Image of 8.7 wt% control fiber: 85% BSA, 15% PEO at magnification of
8.83 x 103 X
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 shown in Figure 4.7 is a clustering of the solution prepared
into the electrospinning machine.
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Figure 4.8: SEM Image of 8.7 wt% test fiber: 33.3% BSA, 33.3% PEO, and 33.3% ampicillin
at magnification of 1.56 x 103 X
In Figure 4.8, 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 shown in Figure 4.8 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; 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 BSA concentration and the PEO concentration have
yielded favorable results; this image shows more distinct fibers that are potentially lined with
19
ampicillin and coated with BSA. Optimally, such a fiber may receive protection from the BSA
coating and remain bioactive from the ampicillin shards.
Figure 4.9: SEM Image of 8.7 wt% test fiber: 33.3% BSA, 33.3% PEO, and 33.3% ampicillin
at magnification of 5.50 x 103 X
Figure 4.9 shows a magnification of a patch of the sample shown in Figure 4.8. 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 the sample shown in Figure 4.7.
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Zone of Inhibition Results (Nanoparticles):
Kanamycin:
Figure 5.1: Results for zone of inhibition test (kanamycin)
T-test conducted
p-value
Difference
Kanamycin VS Surface Adsorption
0.0326
Significant
Kanamycin VS Encapsulation
0.00140
Significant
Surface Adsorption VS Encapsulation
0.00321
Significant
Table 5.1: Summary of T-test conducted within 95% confidence interval
Figure 5.1 show that pure kanamycin has the largest zone of inhibition. Surface
adsorption has a slightly smaller zone of inhibition, whilst encapsulation has the smallest
zone of inhibition.
As shown by Table 5.1, difference in diameter of zone of inhibition between each
sample is significant because p-value of T-test conducted was lower than 0.05. Thus, there
are significant differences between the instantaneous antibacterial effects of samples.
21
Ampicillin:
Figure 5.2: Results for zone of inhibition test (ampicillin)
T-Test conducted
P-value
Difference
Ampicillin VS Surface Adsorption
0.267
Insignificant
Ampicillin VS Encapsulation
0.00254
Significant
Surface Adsorption VS
0.00319
Significant
Encapsulation
Table 5.2: Summary of T-test conducted within 95% confidence interval
Figure 5.2 show that pure ampicillin has the largest zone of inhibition. Surface
adsorption has a slightly smaller zone of inhibition, whilst encapsulation has the smallest
zone of inhibition.
As shown by Table 5.2, difference in diameter of zone of inhibition is only
insignificant between pure ampicillin and surface adsorption because p-value of T-test
conducted was higher than 0.05. Thus differences between the instantaneous antibacterial
effects of samples are only insignificant between pure ampicillin and surface adsorption.
The trend shown in the zone of inhibition test for kanamycin is noted to be reflected
in the zone of inhibition test for ampicillin.
22
Zone of Inhibition Results (Nanofibers):
Figure 6: Picture of treatment against A.tumefaciens using ampicillin loaded nanofibers
Figure 6 is an image of a colony of A.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,
23
such as a fungal contamination. As 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. Furthermore
even the positive control conducted provided no results. A base control solution of 8.7 wt%
85% BSA and 15% PEO was created in which sterile filter paper circles of 0.5 mm radius
was immersed into the solution. An agar cultured with A.tumefaciens was prepared and the
filter paper circles were placed onto the agar in the same formation as shown in 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.
24
Total Plate Count Test Results (Nanoparticles):
Kanamycin:
Figure 7.1: Results for total plate count test (kanamycin)
p-value
T-Test conducted
Day 1
Difference
Day 2
Control VS
0.0168
Insignificant
0.0320
Kanamycin
Control VS Surface
0.00451
Insignificant
0.0336
Adsorbed
Control VS
0.0404
Insignificant
0.0448
Encapsulated
Kanamycin VS
0.983
Significant
0.0342
Surface Adsorbed
Kanamycin VS
0.0431
Insignificant
0.00219
Encapsulated
Surface Adsorbed VS
0.0462
Insignificant
0.00518
Encapsulated
Table 7.1: Summary of t-test conducted within 95% confidence interval
25
Difference
Insignificant
Insignificant
Insignificant
Insignificant
Insignificant
Insignificant
Figure 7.2: Sampling distribution curves for results of total plate count test (kanamycin) on
day 1
Figure 7.3: Sampling distribution curves for results of total plate count test (kanamycin) on
day 2
As shown by Table 7.1, the difference in the number of colonies formed between
control and the 3 samples is significant for both days because p-value of T-Test conducted
was lower than 0.05. Sampling distribution curves shown in Figure 7.2 and 7.3 also shows
minimal overlap between the curves representing the control and the curves representing
the other samples, thus indicating statistically significant differences between samples and
controls on both days. Hence, all samples exhibited antibacterial efficacy.
26
As shown by Figure 7.1, encapsulation has the lowest antibacterial efficacy. Surface
adsorption and pure kanamycin however, had shown to be almost on par in terms of
antibacterial efficacy. In Figure 7.2, the sampling distribution curves showed that there is
huge amount of overlap between the curves representing pure kanamycin and surface
adsorption. Furthermore, the p-values shown in Table 7.1 supported the claim as p-value
was more than 0.05, indicating that on Day 1, pure kanamycin and surface adsorption has
no significant differences in their antibacterial efficacy.
In figure 7.3, the sampling distribution curves showed that the curves representing
pure kanamycin and surface adsorption move apart from each other and decreased the area
of overlap the two graphs has. This shows that on Day 2, antibacterial efficacy of surface
adsorption decreased and is now significantly weaker as compared to pure kanamycin. This
is further backed up by the p-value calculated from the T-test conducted between both
samples, which is less than 0.05 on Day 2, indicating statistically significant differences.
Encapsulation has proved to be the weakest drug delivery technique with significant
differences in antibacterial efficacy when compared to the other samples. This is shown in
Table 7.1 with p-values of less than 0.05, and minimal overlap of encapsulation curves with
curves of other samples as shown in the sampling distribution curves in Figure 7.2 and 7.3.
27
Ampicillin:
Figure 8.1: Results for total plate count test (ampicillin)
p-value
T-Test conducted
Day 1
Difference
Day 2
Difference
Control VS Ampicillin
0.0332
Insignificant
0.0333
Insignificant
Control VS Surface Adsorbed
0.0437
Insignificant
0.0372
Insignificant
Control VS Encapsulated
0.0395
Insignificant
0.0407
Insignificant
Ampicillin VS Surface
Adsorbed
0.529
Significant
0.0386
Insignificant
Ampicillin VS Encapsulated
0.0575
Significant
0.00412
Insignificant
Surface Adsorbed VS
0.0817
Significant
0.0199
Encapsulated
Table 8.1: Summary of t-test conducted within 95% confidence interval
28
Insignificant
Figure 8.2: Sampling distribution curves for results of total plate count test (ampicillin) on day
1
Figure 8.3: Sampling distribution curves for results of total plate count test (ampicillin) on day
2
29
As shown by Table 8.1, the difference in the number of colonies formed between
control and the 3 samples is significant for both days because p-value of T-Test conducted
was lower than 0.05 and Figure 8.2 and 8.3 show sampling distribution curves with minimal
overlap between curves for control and the other samples. Thus there are statistically
significant differences between samples and controls on both days. Hence, all samples
exhibited antibacterial efficacy.
As shown by Figure 8.1, encapsulation remained to have the lowest antibacterial
efficacy. Surface adsorption and pure kanamycin was also almost on par in terms of
antibacterial efficacy. In Figure 8.2, the sampling distribution curves showed again that there
is huge amount of overlap between the curves representing pure kanamycin and surface
adsorption. Furthermore, the p-value shown in Table 8.1 was also more than 0.05, indicating
that on Day 1, pure ampicillin and surface adsorption again has no significant differences in
their antibacterial efficacy.
In figure 8.3, the sampling distribution curves showed that the curves representing
pure ampicillin and surface adsorption move apart from each other and hence area of
overlap decreases. This shows that on Day 2, antibacterial efficacy of surface adsorption
decreased yet again and is now significantly weaker as compared to pure ampicillin. This is
also proved by the p-value calculated from the T-test conducted between both samples,
which is less than 0.05 on Day 2.
Encapsulation has yet again proved to be the weakest with significant differences in
antibacterial efficacy when compared to the other samples. This is shown by p-values of less
than 0.05 in Table 8.1, and minimal overlap of sampling distribution curves as shown in
Figure 8.2 and 8.3.
The trend shown in the total plate count test for kanamycin is noted to be reflected in
the total plate count test for ampicillin.
30
Discussion:
In all tests, drug delivery by surface adsorption is the closest in antibacterial efficacy
as compared to pure antibiotics. This is shown by the high p-values calculated from the Ttests conducted and the huge amount of overlap between sampling distribution curves. This
could be due to the fact that antibiotics are loaded on the surface of the nanoparticles, and
thus surface of particles are similar to that of pure antibiotics. Hence it is almost equivalent to
be using pure antibiotics when drug delivery by surface adsorption is utilized. The decrease
in antibacterial efficacy of surface adsorption by day 2 until it is significantly weaker than
pure antibiotics could also be due to the eroding of the “antibiotic surface” of the
nanoparticles, decreasing the antibacterial efficacy.
Encapsulation remains to be the weakest drug delivery technique. This could be due
to the notion that the drug, being encapsulated within the nanoparticle faces more controlled
drug release. The nanoparticle must first be broken down before the drug can be released.
Thus antibacterial efficacy is severely decreased.
Nanoparticles work as it allows cells to take them up due to its size. This in turn
allows drugs to enter the cytoplasm and increase the pharmacological activity of the drug.
This is important as controlled drug release through the use of nanoparticles reduces the
spread of the drug that will reduce the damage to healthy cells.
The SEM images for nanofibers show that there is very little fibrous construction.
Instead, globs of BSA 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. Addition
31
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. Significant clustering of globs 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. The differences in texture, along with a
comparison with the other control images, led to the conclusion that the fiber in the image
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.
Treatments using nanofibers 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. 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. The notion
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that the strain of bacteria that was being used may be an ampicillin-resistant strain was also
considered. Several 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, the notion
that the growth rate exceeded the effects of the antibiotic was eliminated. 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. Separate sets of filter paper were soaked in both solutions, and placed 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.
Limitations:
We are unable to ensure bioactivity of antibiotics remains during the formation of
nanoparticles and drug loading, thus the total amount of antibiotics that are still active may
not be the same between samples. Hence, we minimized the amount of time the antibiotics
spend in room conditions, and when not in use, the solution containing the antibiotics will be
placed in the refrigerator at 4oC immediately.
Future plans:
33
For our project, it can be extended to cultivating A.tumefaciens on potato cylinders,
and be subjected to treatment by antibiotic-loaded nanoparticles or nanofibers. Here, we can
investigate the practicality and feasibility of our nanoparticles and nanofibers to be used as a
more effective treatment as we work towards using live plants as subjects for treatment.
Considering success of nanoparticle surface adsorption, a new nanofiber method in
which fiber is immersed into antibiotic solution similar to surface adsorption will be tested.
Research also indicates that nanoparticles and nanofibers, 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 and nanoparticles by manipulating them with such fields,
thus increasing their overall efficiency.
Conclusions:
In conclusion, nanofibers still hold promise in integrating ampicillin sodium salt and
bovine serum albumin into a feasible drug delivery system. 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.
In our context, drug loaded nanoparticles are thus more effective than drug loaded
nanofibers for antibacterial properties, and surface adsorption was the most effective
developed technique for drug delivery.
Application:
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Our antibiotic loaded nanoparticles and nanofibers can be incorporated into an
aerosol spray, and be used for a more effective treatment for A.tumefaciens infected plants,
reducing environmental impact and be more economically conducive.
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Acknowledgement
We would like to thank our mentor, Ms Jennifer Ting Jeum Shian and Mr Jason Beltz
for their guidance and mentoring throughout the project. We would also like to thank Mr
George Wolfe, Mr Duke Writer and Mdm Lim Cheng Fui for assisting us during our project.
We are also grateful to our friends, seniors, teachers and any other people who have
assisted us in any way.
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