Nanoparticle Toxicity Assessment in a Bacterial Model Ian L. Gunsolus, Dehong Hu, Cosmin Mihai, William B. Chrisler, Samuel E. Lohse, Marco D. Torelli, Robert J. Hamers, Catherine J. Murphy, Galya Orr, and Christy L. Haynes November 4, 2013 NSF Center for Sustainable Nanotechnology
Iterative Approach Determine the functional impact of nanoparticles on biological and ecological model systems QDots S. oneidensis 10-15 nm ZnS shell CdSe core -NH 3 + or -COO - TiO 2 Nanoparticle-Induced ROS Correlates with Modulated Immune Cell Function, Maurer-Jones, M.A., Christenson, J. R., and Haynes, C.L. J. Nano. Res., 14 1291-1303 (2012).
Model Bacterial System: Shewanella oneidensis MR-1 found world-wide survives in a variety of environments reduces metals and metal oxides in geochemical nutrient cycling *S. oneidensis was a generous gift from J. Gralnick (UMN Microbiology)
Iterative Approach Determine the functional impact of nanoparticles on biological and ecological model systems QDots S. oneidensis Ideally: High resolution Live cells Robust labeling TiO 2 Nanoparticle-Induced ROS Correlates with Modulated Immune Cell Function, Maurer-Jones, M.A., Christenson, J. R., and Haynes, C.L. J. Nano. Res., 14 1291-1303 (2012).
Super-Resolution Fluorescence Microscopy In this work, we used both: stochastic optical reconstruction microscopy (STORM) and structured illumination microscopy (SIM) Image of two spots in a light microscope At times when only the left or right spot is on", we find the center We plot just the centers for a STORM image Need fluorescent bacteria and fluorescent nanoparticles For STORM, fluorophores must be photoswitchable For SIM, fluorophores must be photostable and have high QY Achieve optical spatial resolution as low as 20 nm (STORM) or 120 nm (SIM)
Staining Bacteria Cell Surfaces for Super-Resolution Studies Amine-reactive AlexaFluor 488 (a bright, photostable, and photoblinking dye) 2 hours Fluorescently-tagged primary amine Primary amine in biomolecule (e.g. protein, lipopolysaccharide, or peptidoglycan to highlight outer membrane) Super-resolution image of S. oneidensis MR-1 stained with amine-reactive dye
Imaging Gram-Negative Bacteria Top: wide-field images of S. oneidensis MR-1 Bottom: Corresponding STORM images
Imaging Gram-Positive Bacteria Laser-scanning confocal image of B. subtilis 168 Structured-illumination microscopy image of B. subtilis 168
Imaging Cell-Nanomaterial Interactions Traditional imaging methods: limited information on nanomaterial localization patterns with bacterial cells Laser-scanning confocal image of S. oneidensis MR-1 associated with Qdot 605 ITK Amino (PEG) Quantum Dots (Life Technologies)
Imaging Cell-Nanomaterial Interactions 1 unit = 420 nm Structured illumination microscopy image of S. oneidensis MR-1 associated with Qdot 605 ITK Amino (PEG) Quantum Dots (Life Technologies). Qdots localize at the cell surface but are not internalized
How do Nanoparticles Interact with Bacterial Cells? Extracellular environment Interface Intracellular environment Hypothesis: Nanoparticles bind lipopolysaccharides (LPS) and outer-membrane proteins (OMP) and localize at the cell surface. Figure: http://www.studyblue.com/
Compare Quantum Dot Interactions Qdot 10-15 nm ZnS shell CdSe core + EDTA -NH 3 + or -COO - both before and after removal of lipopolysaccharides and outer-membrane proteins. Figure: http://www.studyblue.com/
Monitoring Protein and LPS Depletion EDTA treatment removes protein and ~50% of the LPS from the cell surface
Visible Qdot association with intact LPS, OMP No visible Qdot association after LPS, OMP removal Before LPS, OMP Removal Qdot After LPS, OMP Removal Qdot
Quantifying Cell-Qdot Binding using Flow Cytometry 0 nm Qdot 250 nm Qdot Cells with associated Qdots Cells alone New population (with high SSC and fluorescence at Qdot emission) contains nucleic acids Qdot + cells.
Flow Cytometry-Based Quantitation of Cell-Qdot Binding 0 mm EDTA 5 mm EDTA 250 nm Qdot LPS, OMP removal (EDTA treatment) may decrease Qdot binding to S. oneidensis MR-1 Electrostatic attraction may influence this interaction
Qdot Effect on Cell Membrane Integrity Non-significant increase in membrane permeability following Qdot exposure (regardless of Qdot ligand).
Conclusions We ve developed a simple, off-the-shelf staining protocol to facilitate bacteria super-resolution imaging Quantum dots associate with, but do not penetrate, the model gram-negative and gram-positive bacterial strains Lipopolysaccharides and/or outer membrane proteins may mediate bacteria-qdot association Associated Qdots (at the concentrations probed) do not compromise S. oneidensis membrane integrity
Acknowledgements www.sustainable-nano.com http://susnano.chem.wisc.edu/ Center for Sustainable Nanotechnology