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 = grams 2. Nisin: 0.1 M solution = 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 #FilterTreatmentZone Size (mm) 3A M Col; 0.01 M Amp41 3B M Col; 0.1 M Nisin48 3C M Col; (30 uL) 0.01 M Amp; 10 uL E. coli0 3D M Col; (30 uL) 0.10 M Nis; 10 uL E. coli M Ampicillin H20H200 4A M Col; 0.01 M Amp; 15 mL Blood41 4B M Col; 0.1 M Nis; 15 mL Blood26 4C M Col; 0.01 M Amp; 10 mL E. coli55 4D M Col; 0.1 M Nis; 10 mL E. coli M Amp H20H200

Data: Most Successful Patches Day 1Filter SizeTreatmentZone Size 1C M Col; 0.10 M Nis55 2C M Col; 0.01 M Amp56 2D M Col; 0.01 M Nis52 Day 2Filter SizeTreatmentZone Size 3D M Col; 30 µL 0.10 M Nis; 10 µL E. coli55 4C M Col; 0.01 M Amp; 10 µL E. coli; 15 µL blood55 Day 3Filter SizeTreatmentZone Size 6D M Col; 10 µL E. coli; 20 µL 0.10 M Nis36 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 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 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 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 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.

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