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 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. 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 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. Hypothesis The two delivery techniques will be comparable, through both qualitative and quantitative means Variables Independent • Method of drug delivery Dependent • Efficacy of drug delivery system Controlled / constant • Type of bacteria (A.tumefaciens) • Volume and types of antibiotic - tetracycline and ampicillin • Agrobacterium volume • Sizes of potato strips • Temperature and humidity Equipment Electrospinning apparatus Scanning electron microscope (SEM) Homogenizer Incubator Environmental chamber Spectrophotometer Materials • • • • • • Bovine Serum Albumin Alcohol A. tumefacians Potato strips Diffusion assays tetracycline and ampicillin 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 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 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 Antibiotic loading - nanofibres Antibiotics mixed in albumin solution Homogenous solution Hypothesis that spinning solution will result in the non polymer antibiotics also being spun Effectiveness of antibioticloaded nanoparticles • • • • Protein nanoparticles digested by proteases to release antibiotics Antibiotic-loaded nanoparticles are subjected to: A.tumefacians agar plates discs A.tumefacians-potato strips Timeline (HCI) Form droplets w/ specific concentration and temp. Examine results and modify original solution Send for characterization Load droplets with antibiotics Test droplets Timeline (AOS) Form solution with specific concentration Examine results and modify original solution Send for characterization Spin solution Test fibers 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), 553562 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 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. Knee, M., & Nameth, S. (2007). Horticulture and Crop Science: Bacteria. The Ohio State University, Horticulture Department. Retrieved September 12, 2010 from : http://www.hcs.ohiostate.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 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), 19181923.