1. Enhancing Antibacterial Efficacy using Protein Nanoparticles Leslie Tan Zheng Yu Tan Jing Chong Erik Warnquist Varun Kulkarni Retrieved from: http://www.eng.u ci.edu/files/images /gallery/Protein_N anoparticle_Struct ure.jpg

2. Introduction  Pesticides are used to eradicate Agrobacterium tumefaciens  High percentage of pesticide does not reach the target species.  Result in water and soil pollution.  Threatens biodiversity.

3. Introduction  Usage of nanoparticles as drug carrier for pesticides  Increase in therapeutic efficacy  Increasing localisation to diseased sites  Decrease in side effect  Protein Nanoparticle are biodegradable, metabolisable and non-antigenic  Does not accumulate in tissue

4. Objective  To compare the effectiveness of antibiotic loaded albumin nanodroplets against antibiotic loaded albumin nanofibre on A. tumefaciens, grown both in vitro and in vivo.

5. Hypothesis  The two delivery techniques will be comparable, through both qualitative and quantitative means

6. Variables Method of drug delivery Independent Efficacy of drug delivery system Dependent Type of bacteria (A.tumefaciens) Volume and types of antibiotic - tetracycline and ampicillin Agrobacterium volume Sizes of potato strips Temperature and humidity Controlled / constant

7. Equipment  Electrospinning apparatus  Scanning electron microscope (SEM)  Homogenizer  Incubator  Environmental chamber  Spectrophotometer

8. Materials Bovine Serum Albumin Alcohol A. tumefacians Potato strips Diffusion assays tetracycline and ampicillin

9. Preparation of albumin nanodroplets Emulsification Aqueous Bovine Serum Albumin is turned into an emulsion at room temperature and in oil A homogenizer is used to make the emulsion homogeneous. There is a high dispersion of particles Emulsion is added to pre-heated oil Albumin nanoparticles are separated by desolvating agent eg. Alcohol

11. Preparation of albumin nanofibers Electrospinning Solution inside a syringe exposed to initial electric field Electric field increases in charge Point is reached where attractive forces of charges exceeds surface tension The fibers are projected onto a grounded collector

12. Antibiotic loading - nanodroplets Incubating nanoparticles in antibiotic solution Antibiotic contained in nanoparticles Done at protein's isoelectric point  Minimum solubility and maximum absorption  BSA: pH of 4.4 Larger amount of antibiotic loaded Antibiotic entrapment efficacy measured

13. Antibiotic loading - nanofibres  Antibiotics mixed in albumin solution  Homogenous solution  Hypothesis that spinning solution will result in the non polymer antibiotics also being spun

14. Effectiveness of antibiotic- loaded nanoparticles Protein nanoparticles digested by proteases to release antibiotics Antibiotic-loaded nanoparticles are subjected to: A.tumefacians agar plates discs A.tumefacians-potato strips

15. Timeline (HCI) Form droplets w/ specific concentration and temp. Load droplets with antibiotics Test droplets Send for characterization Examine results and modify original solution

16. Timeline (AOS) Form solution with specific concentration Spin solution Test fibers Send for characterization Examine results and modify original solution

17. References  Buschle-Diller, G., Cooper, J., Xie, Z., Wu, Y., Waldrup, J., & Ren, X. (2007). Release of antibiotics from electrospun bicomponent fibers. Cellulose, 14(6), 553- 562  Collins, A. (2001). Agrobacterium tumefaciens. Department of Plant Pathology, University of North Carolina State. Retrieved September 19, 2010 from: http:/www.cals.ncsu.edu/course/pp728/ Agrobacterium/Alyssa_Collins_profile.htm

18.  Frenot, A., & Chronakis, I.S. (2003). Polymer nanofibers assembled by electrospinning. Current Opinion in Colloid and Interface Science, 8(1), 64-75.  Hyuk, Y.S., Taek, G.K., & Park, T.G. (2009). Surface-functionalized electrospun nanofibers for tissue engineering and drug delivery. Advanced Drug Delivery Reviews, 61(12), 1033-1042.  Jahanshahi, M. & Babaei, Z. (2008). Protein nanoparticle: A unique system as drug delivery vehicles. African Journal of Biotechnology, 7(25), 4926-4934.

19.  Knee, M., & Nameth, S. (2007). Horticulture and Crop Science: Bacteria. The Ohio State University, Horticulture Department. Retrieved September 12, 2010 from : http://www.hcs.ohio- state.edu/hcs300/bact.htm  Kratz, F. (2008). Albumin as a drug carrier: Design of prodrugs, drug conjugates and nanoparticles. Journal of Controlled Release, 132(3), 171-183.  McManus, P. (2007). Antibiotic Use in Plant Disease Control. Fruit Pathology: University of Wisconsin-Madison. Retrieved September 13, 2010 from: http://www.plantpath.wisc.edu/fpath /antibiotic-use.htm

20.  M.R., Jahanshahi, M., & Najafpour, G.D. (2006). Production of biological nanoparticles from bovine serum albumin for drug delivery. African Journal of Biotechnology, 5(20), 1918- 1923.