Transcript Abstract - Oregon State University

Abstract
This experiment was done to test the efficiency of
transdermal patches in delivering lantibiotics through the
surface of a membrane by diffusion. We accomplished
this by simulating cell layers of human skin. Using .22
and .45 µm (micron) filters as “patches,” we coated each
with a different combination of materials. The materials
used were Collagen, Ampicillin, Nisin, horse blood, and
E. coli. Placing the patches on agar plates, which were
seeded with Pediococcus a Gram positive bacteria
(bacteria with thicker cell walls), we identified the patch
that had best drug delivery by looking at how many
bacteria around the patch were killed. This area of
clearing is called a zone.
Introduction
Transdermal patches have gained popularity
in delivering drugs like birth control, nicotine,
nitroglycerin. This method of treatment is more
advantageous because of the fact that the delivery
process is painless and easier to use. The purpose
of our experiment was to test the efficiency of
transdermal patches in the delivering of lantibiotics
through the surface of a membrane. In doing so,
we find which patch works the best in transporting
lantibiotics.
Words to Know
• Buffer: a solution that can keep its relative acidity or pH constant,
despite the addition of strong acids or strong bases.
• Diffusion: the spontaneous migration of substances from an area
of high concentration to an area of lower concentration
across a membrane.
• E. coli: common bacterium that normally inhabits the intestinal
tracts of humans and animals and is Gram-negative.
• Nisin: a natural antimicrobial agent used as a lantibacterial, and
is several orders of magnitude larger than ampicillin
• Pediococcus: gram-positive bacteria (thicker membranes)
• Collagen: a large molecule that is made up of fibers that is a
principle component of skin and bones.
• Ampicillin: A semisynthetic penicillin having a broader
antibacterial spectrum of action that than of penicillin (gram
negative and positive).
Components and Procedures
• PBS (Phosphate Buffer of pH 7.4): used for making of
solutions
• Solvent Solutions (simulating medication):
1. Ampicillin: 0.01 Molarity solution = 0.1857 grams
•
•
2. Nisin: 0.1 M solution = 0.125 g; 0.01 M solution = .0125g
3. Gelatin: 0.1 M = 0.5g; for 0.01 M = 0.05g
Bioassay Plates: test media for biological efficiency of
patch
Patches: simulate painless drug delivery due when coated
with collagen and solvent solutions that represent the
drugs
• Collagen: matrix to contain proteins
• E. coli: bacteria that simulates bacteria in human skin.
Data: Day 2 (Dishes 3, 4 and 5)
Patch #
Filter
Treatment
Zone Size (mm)
3A
0.45
0.10 M Col; 0.01 M Amp
41
3B
0.45
0.10 M Col; 0.1 M Nisin
48
0.45
0.10 M Col; (30 uL) 0.01 M
Amp; 10 uL E. coli
0
3D
0.45
0.10 M Col; (30 uL) 0.10 M
Nis; 10 uL E. coli
55
3+
0.45
.01 M Ampicillin
50
3-
0.45
H20
0
0.22
0.10 M Col; 0.01 M Amp;
15 mL Blood
41
0.22
0.10 M Col; 0.1 M Nis;
15 mL Blood
26
0.45
0.10 M Col; 0.01 M Amp;
10 mL E. coli
55
4D
0.45
0.10 M Col; 0.1 M Nis;
10 mL E. coli
43
4+
0.45
0.01 M Amp
37.5
4-
0.45
H20
0
3C
4A
4B
4C
Data: Most Successful Patches
Day 1
Filter Size
Treatment
Zone Size
1C
0.22
0.10 M Col; 0.10 M Nis
55
2C
0.45
0.10 M Col; 0.01 M Amp
56
2D
0.45
0.10 M Col; 0.01 M Nis
52
Day 2
Filter Size
Treatment
Zone Size
0.45
0.10 M Col; 30 µL 0.10 M Nis;
10 µL E. coli
55
4C
0.45
0.10 M Col; 0.01 M Amp;
10 µL E. coli; 15 µL blood
55
Day 3
Filter Size
Treatment
Zone Size
0.45
0.10 M Col; 10 µL E. coli;
20 µL 0.10 M Nis
36
3D
6D
These are the patches with the largest zones from each day of testing. The data
shows that 0.10 M Collagen and 0.01 M Ampicillin work well. 0.10 M Nisin also
works. The E. coli that was not resistant to Ampicillin (Day 2) worked better than
the E. coli that was (Day 3). .45 micron filters are the best.
Ampicillin vs. Nisin as Lantibiotics
Comparing Ampicillin and Nisin
60
Zone Size
50
40
30
20
10
0
0.10 M Col; 0.01 0.10 M Col; 0.10 0.10 M Col; 0.01
M Amp
M Nis
M Amp
0.22
0.22
0.45
0.10 M Col; 0.1
M Nisin
0.45
Treatment
 In the patches with only collagen and a lantibiotic, nisin created a larger zone
than ampicillin, regardless of the filter size. However, when observing the
zones, those created by ampicillin are more obvious. This graph is misleading
because though nisin spreads further, ampicillin may kill more bacteria.
Results: Plate #6
Zone Size
Comparison of Plates with Ampicillin-resistant E. coli
40
35
30
25
20
15
10
5
0
0.10M Col; 10 µL E. 0.10M Col; 10 µL E. 0.10M Col; 10 µL E. 0.10M Col; 10 µL E.
coli;
coli;
coli;
coli;
20 µL 0.01M Amp
20 µL 0.10M Nis
20 µL 0.01M Amp
20 µL 0.10M Nis
0.22
0.22
0.45
0.45
Treatment
This graph displays our results from a single plate of patches
coated with ampicillin-resistant E. coli. The comparison of
ampicillin and nisin differs from that found in other plates.
Results: Different Types of E. coli
Comparison of Normal E. coli and Ampicillinresistant E. coli
45
40
35
Zone Size
 This graph
compares the
effects of normal
E. coli and E.
coli that was
subcultured in
ampicillin and
so is resistant to
ampicillin. The
zone size of a
patch with
collagen and
ampicillin only is
included as a
reference.
30
25
20
15
10
5
0
0.10 M Col;
0.01 M Amp
0.10 M Col; 10 0.10 M Col; 10 0.1 M Col; 10 0.10 M Col; 10
µL E. Coli; 30 µL *E. Coli*;
µL Blood; 10 µL Blood; 10
µL 0.01 M
20 µL 0.01 M µL E. Coli; 20
µL *E. Coli*;
Amp
Amp
µL 0.01 M
20 µL 0.01 M
Amp
Amp
Treatment
Results: An Overview
 The majority of the patches with larger zones used .45 µm filters
 Nisin consistently created a larger zone diameter than ampicillin.
However, the zone made by ampicillin was clearer, and that of
nisin was shaped so that the measurement was not entirely
accurate. Nisin is a larger molecule so it is conceivable that it
did not penetrate as much of the membranes but it found
another way to spread out.
 The subcultured E. coli worked better when only collagen and
ampicillin was included, but normal E. coli worked better when
horse blood was added. No E. coli at all worked the best, but E.
coli was used in the study to imitate the cell layers of skin.
 Horse blood, also used to mimic the cell layers of the skin, was
relatively successful, especially together with ampicillin.
 0.1 M Collagen, 0.01 M Ampicillin, and 0.1 M Nisin are the most
successful dilutions of these solutions
Conclusion
After having experimented with different combinations
of solutions to coat the patches, we have discovered that our
early hypothesis was incorrect. We thought that the
Ampicillin was going to spread furthest in the agar plates
because it is a smaller molecule than Nisin. Nisin proved to
be less effective at killing all the surrounding bacteria.
However, it spread further lengthwise. We also found that
.45 µm filters allowed the lantibiotics to diffuse more
efficiently, since they have larger pores.
To improve our experiment in the future, things we
could do would be to try to find more precise ways to model
the layers of skin, to allow more time for data collection, and
to develop a better method for measuring the strength of the
drugs coated on the patches.
Acknowledgements
We would like to thank Iva Jovanovic for
being our mentor for this project and being here
to help us with and explaining the different
concepts involved in this process. Also, Drs.
Michelle Bothwell and Joe Mcguire for
generously allowing us to use their lab, Dr. Skip
Rochefort for giving us incredible guidance and
support. Last but not least, we’d like to thank all
the people at SESEY who have made this such
a great experience for us! Thanks much,
Miranda Fix and Michelle Zhao