Presentation on theme: "PEGylated Gold Nanoparticles Conjugated to"— Presentation transcript:
1PEGylated Gold Nanoparticles Conjugated to Monoclonal F19 Antibodies as TargetedLabeling Agents for Human Pancreatic Carcinoma TissueFebruary 26, 2009Gary CraigDepartment of Chemical and Biological EngineeringUniversity of Maine
2Sections of Talk 1. Pancreatic Cancer 2. Noble Metal Nanoparticles 3. Functionalization and Bio- Linking4. Tissue Studies5. Conclusions6. Future Directions
3Application to Pancreatic Cancer Detection Pancreatic Cancer produceslow density tumor masses:Not visible early by screeningmethods (CT scanning)During surgical removal, thetumor margins are difficult tofind.Early diagnosis – whole animal imaging (x-ray tomography)Help the surgeon identify cancer margins during surgery.
4Histology of Pancreatic Adenocarcinoma (Standard Diagnostic Method) Standard Histology StainLabeled for Cancer StromaHealthy Pancreatic Tissue
5Histology of Pancreatic Adenocarcinoma Standard Histology StainCancer Stroma LabeledPancreatic Cancer Tissue
6Interconnected Themes of Interest in our Lab Observation of (Bio)-Chemical processes at the single-molecule level in heterogeneous systems(1)Sample Driven Design Constraints:Question (material or animal system)GeometryChemical/Physical EnvironmentTime scale(3)Measurement(Tissue)Nanoprobe Photophysics – Understanding Chemical and Physical characteristics(2)Developing nanoprobes for imaging at the single molecule level (Engineering and synthesis)Development of imaging tools for single molecule detection
7We are Engineers Design Requirements 1. Small, shape and size tunable 2. Non-toxic3. Biocompatible (stable, non-aggregating).4. Long-circulating in blood – not rapidlyremoved by the body5. Target Specific (sticks only to “cancer”)6. Significant improvement in image contrast7. Cost effective (health care $$$)We are Engineers
8Metallic Nanoparticles – Batch Synthesis Metal salts are brought to boil in aqueous solution (HAuCl4.H2O for gold)A reducing agent is added (sodium citrate) and reaction proceeds for 2 minutesExcess reducing agent electro-statically stabilizes against aggregation100 oCOstwaldRipeningStirringtimeAu3+Au0ReducingStableColloidSize depends on the molar ratio of metal ion and reducing agentRod shaped particles formed via surfactant templating (CTAB, TOAB)
9Metallic Nanoparticles Gold(spheres)Gold(Rods)We are interested in designing our own nanoprobes, to have engineered properties which enhance that capabilities of our imaging technique. This process is already underway in our group and part of a larger IMB effort.Photo-physics??Size and geometry tunable (2.5 – 65 nm)Very expensive to buy (spheres), relatively cheap and easy to make
11Particle Stability Ideally particles should not agglomerate Typical gold synthesis produces a stable colloid, but not stable enough to be used in vivoChanges in temperature, pH, concentration will cause agglomeration
12Part I. Simple Light Scattering Elastic Scattering (Rayleigh) – energy of the photon is unchangedA light induced oscillation of conduction electrons gives rise to a peak in the absorption curve (Plasmon Resonance).Small particles scatter shorter wavelengths,Larger particles scatter longer wavelengths
15Surface ModificationCovalently bonding a polymer to the surface of the particle sterically prevents agglomerationThiol linked polyethylene glycol (PEG) is convenient for gold particlesProvides point ofattachment oftargeting moleculePolyethylene Glycol Dithiol(Wolfgang Eck)
17Antibodies Proteins produced by the immune system Used to target foreign material such as bacteria or virusesConvenient targeting mechanism
18Monoclonal Antibody F19Developed at the Ludwig Institute for Cancer ResearchSpecific to FAP α (expressed in stroma)Coupled to PEG via NHS/EDC chemistryPurified by Size Exclusion Chromatography
19Y Y Y Y Y Y Design Approach mAb F19 functionalized gold nanoparticles for visualizationof pancreatic cancer stromal cellsYYYYYYYThe PEG coating enhances particle biocompatibility and eliminates agglomeration (Electro-static and Steric).Antibodies can be attached via the terminal carboxy groups using standard NHS/EDC coupling chemistry.F-19 antibody coupled via the carboxy moiety (Ludwig Institute) – Binds to glycoproteins on stromal cell surface
20PEG Dithiol SynthesisSynthesizing our own allows us to control length and functionality
21Gold Nanoparticles Narrow size distribution Broad size distribution (40 mm images)
22SEC Column FractionsThe antibody-functionalized gold nanoparticles can be fractionated according to size and antibody content by size exclusion chromatography (SEC). They can be prepared free from any non-bound antibody and are fully stable over a period of at least several weeks.
24NIH 3T3 Cells Labeled with WGA conjugated Gold Nanoparticles
25Cancer 40x + Au-PEG - Control Cancer 40x + Au-IgG ControlB35 mmA35 mm
26A. B. Comparison of stained vs Au-mABF19 lableled 35 mm35 mmA.B.Histological StainingLabeling with F19 Conjugate
27Cell / Ex Vivo labeling - Preliminary < 5 mm pancreatic tissue sectionsNegativecontrolImmunohistochemicallyStainedF-19 NanoparticleLabeledOptimization of nanoparticle geometry for optical contrastReproducibilityLots of tissue studiesNanotoxicity (John Wise – USM)
28Immunoelectron Microscopy Gold Scatters Electrons!Labeling with ConjugatesFixation and EmbeddingViewed by TEMLabeling or Tissue Expression?
29Conclusions:Gold nanoconjugates have great potential in cancer diagnosis and treatment due to their optical contrastPegylated gold is extremely stableMAB-F19 based conjugates show promise but more characterization is necessary.
30Future Directions Hard to get F19 Problems with animal model – Different antibody?Other contrast mechanisms-CT Contrast
31ReferencesW J Rettig, P Garin-Chesa, H R Beresford, H F Oettgen, M R Melamed, and L J Old. Cell-surface glycoproteins of human sarcomas: differential expression in normal and malignant tissues and cultured cells. Proc Natl Acad Sci U S A May; 85(9): 3110–3114.W. Eck, G. Craig, A. Sigdel, G. Ritter, L. Old, L. Tang, M. Brennan, P. Allen and M. Mason, PEGylated Gold Nanoparticles Conjugated to Monoclonal F19 Antibodies as Targeted Labeling Agents for Human Pancreatic Carcinoma Tissue. ACS Nano, 2(11): , November 2008.G.D. Kymionus, M.M. Konstadoulakis, E. Leandros, A. Manouras, A. Apostolou, D. Alexiou, S. Katsaragakis and G. ANDROULAKIS. J.R. Effect of Curative versus palliative surgical treatment for stage III pancreatic cancer patients . Coll.Surg.Edinb., 44, August 1999,
32The Mason Group Thank You! Gary CraigMatt KingRay KennardEd AllgeyerDavid PaulSara SterlingBen FreedmanSushil KadkaAruna SigdelMike BrowneAdam PonganEric YoungJennifer BrownEben EstellMaria CollinsCollaboratorsWilliam DeSistoSam HessDavid NeivandtPeter AllenWolfgang EckThank You!Support: NSF, Memorial Sloan Kettering Cancer Center
33Excessive intake of Silver colloids (Argyria) Questions?