Association of Gold Nanoparticles with Bacterial Lipopolysaccharides

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1 Association of Gold Nanoparticles with Bacterial Lipopolysaccharides Kurt H Jacobson, Samuel E Lohse, Julianne Troiano, Franz M Geiger, Catherine J Murphy, Joel A Pedersen University of Wisconsin Madison Center for Sustainable Nanotechnology

2 Nanoparticle Interaction with Bacterial Surfaces Bacterial cells are expected to interact with NPs in a wide variety of environments: Wastewater treatment Soils Plants In host organisms Structured illumination fluorescence microscopy image of nanoparticle (red) interaction with bacterial outer membrane components (green). Squares in background are 1.2 µm 2. How do nanoparticle size, aspect ratio and surface chemistry affect attachment to, penetration of, and disruption of cellular membranes? Gunsulos et al. (in prep.)

3 Gram Negative Bacteria Escherichia coli Klebsiella Salmonella Vibrio 200 nm Outer Membrane Inner Membrane Lipopolysaccharide Cyanobacteria (LPS) Pseudomonas Rhizobium Periplasmic Shewanella space Adapted from Brinkmann et al. PLoS Biol Adapted from Raetz & Whitfield. Annu. Rev. Biochem. 2002

4 Lipopolysaccharide (LPS) - - n O-antigen Not always present Rough LPS lacks O-antigen Smooth LPS contains O-antigen Variable length, n = 0 50 Composition varies by species and strain Low abundance sialic acid (pk a < 3) and galacturonic acid (pk a < 5) residues Core Oligosaccharide Composition varies by species and strain 2 5 phosphate groups (pk a < 3) Lipid A Hydrophobic companion to inner leaflet phospholipids Little chemical variation between species Responsible for much of the toxicity of LPS Adapted from Erridge et al. Microbes Infect. 2002

5 Objectives Develop in-situ, versatile platforms for studying the interactions of engineered NPs with LPS Investigate the degree to which LPS influences the interactions of NPs with bacterial outer membranes Test the hypothesis that NP LPS interactions are governed by electrostatics

6 Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D) f ( manalyte + mwater) D stiffness For rigid films: Dm = -C QCM Df n n m = change in mass n = overtone number fn = change in frequency C QCM = mass sensitivity factor In situ, label-free monitoring of bilayer formation and interaction with NPs 6

7 f 5 /5 (Hz) D (10-6 ) Supported Lipid Bilayer (SLB) Formation

8 n Smooth LPS from Salmonella (slps) Incorporation of LPS into Lipid Vesicles 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine POPC (16:0 18:1 PC) Sonication + extrusion through 50 nm membrane filter nm

9 Incorporation of LPS into Lipid Vesicles 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine POPC (16:0 18:1 PC) Sonication + extrusion through 50 nm membrane filter nm Rough LPS from Salmonella (rlps)

10 Vesicle Electrophoretic Mobility as Function of LPS Content I = 1 mm (NaCl), ph 7.4 (2 mm HEPES)

11 Formation of LPS-Containing Bilayers Smooth LPS Rough LPS areal mass density (ng cm -2 ) composition mean SD 0% slps % slps % slps areal mass density (ng cm -2 ) composition mean SD 0% rlps % rlps % rlps 474 4

12 ~1 nm ~5 nm ~2nm ~15 40 nm Supported bilayers containing LPS SiO 2 SiO 2 SiO 2 100% POPC Smooth LPS doped Rough LPS doped Strauss et al., J. Mol. Recognit., 2009

13 Functionalized Gold Nanoparticles (AuNPs) Synthesized using a modified Schiffrin procedure 20 nm Primary particle size = nm Lohse et al. ACS Nano, 2013 Brust et al. J. Chem Soc. Chem. Comm., 1994

14 AuNP Ligands Negatively charged Positively charged Mercaptopropionic Acid (MPA) Mercaptopropyl Amine (MP-Amine) Ionic Strength (mm) d h (nm) ζ-potential (mv) MPA AuNPs 13 ± 5 68 ± ± ± 3-27 ± 6-25 ± 3 MP-Amine AuNPs 12 ± 4 57 ± ± ± 4 29 ± 2 26 ± 8

15 Mass of NP Attachment Representative QCM-D Trace AuNP with Bilayer NPs Rinse 12.8 nm MP-Amine AuNPs to 100% POPC bilayer, I = 25mM, ph7.4

16 Attachment of MPAmine-AuNPs Smooth LPS Rough LPS MPAmine-AuNP attachment increases as % LPS increases as ionic strength decreases for rough vs. smooth LPS

17 Conclusions & Future Directions Interactions between NPs and LPS are dominated by electrostatic forces Gram-negative bacteria exposed to positively charged NPs are expected to accumulate particles in their LPS layer Bacteria with Rough LPS will accumulate more particles Super-resolution microscopy to image accumulation of NPs in LPS-containing bilayers In situ Raman spectromicroscopy to track molecular level interactions between NPs and LPS molecules Non-linear spectroscopy to study changes in lipid order

18 Acknowledgments Centers for Chemical Innovation Center for Sustainable Nanotechnology CHE Joel Pedersen Tom Kuech Eric Melby Cathy Murphy Sam Lohse Christy Haynes Ian Gunsolus Bob Hamers Lee Bishop Marco Torelli Arielle Mensch Franz Geiger Julianne Troiano Laura Olenick Gayla Orr

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