PEGylated Gold Nanoparticles Conjugated to Monoclonal F19 Antibodies as Targeted

1. PEGylated Gold Nanoparticles Conjugated to Monoclonal F19 Antibodies as Targeted Labeling Agents for Human Pancreatic Carcinoma Tissue February 26, 2009 Gary Craig Department of Chemical and Biological Engineering University of Maine

2. Sections of Talk • 1. Pancreatic Cancer • 2. Noble Metal Nanoparticles • 3. Functionalization and Bio-Linking • 4. Tissue Studies • 5. Conclusions • 6. Future Directions

3. Application to Pancreatic Cancer Detection Pancreatic Cancer produces low density tumor masses: Not visible early by screening methods (CT scanning) During surgical removal, the tumor margins are difficult to find. Early diagnosis – whole animal imaging (x-ray tomography) Help the surgeon identify cancer margins during surgery.

4. Histology of Pancreatic Adenocarcinoma (Standard Diagnostic Method) Standard Histology Stain Labeled for Cancer Stroma Healthy Pancreatic Tissue

5. Histology of Pancreatic Adenocarcinoma Standard Histology Stain Cancer Stroma Labeled Pancreatic Cancer Tissue

6. Interconnected Themes of Interest in our Lab Observation of (Bio)-Chemical processes at the single-molecule level in heterogeneous systems Sample Driven Design Constraints: Question (material or animal system) Geometry Chemical/Physical Environment Time scale (1) (3) Measurement (Tissue) NanoprobePhotophysics – 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

7. Design Requirements 1. Small, shape and size tunable 2. Non-toxic 3. Biocompatible (stable, non-aggregating). 4. Long-circulating in blood – not rapidly removed by the body 5. Target Specific (sticks only to “cancer”) 6. Significant improvement in image contrast 7. Cost effective (health care $$$) We are Engineers

8. Metallic 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 minutes • Excess reducing agent electro-statically stabilizes against aggregation 100 oC Ostwald Ripening Stirring time Au3+ Au0 Reducing Stable Colloid • Size depends on the molar ratio of metal ion and reducing agent • Rod shaped particles formed via surfactant templating (CTAB, TOAB)

9. Metallic Nanoparticles Gold (spheres) Gold (Rods) Photo-physics?? Size and geometry tunable (2.5 – 65 nm) Very expensive to buy (spheres), relatively cheap and easy to make

10. Systems Under Investigation Metals: Silver Nitrate (AgNO3) Gold Acid Chloride (HAuCl4) Reducing Agents: Sodium citrate (mild) Sodium borohydride(strong) Surfactants: Cetyltrymethylammonium Bromide (CTAB) Polyethylene Glycol

11. Particle Stability • Ideally particles should not agglomerate • Typical gold synthesis produces a stable colloid, but not stable enough to be used in vivo • Changes in temperature, pH, concentration will cause agglomeration

12. Part I. Simple Light Scattering Elastic Scattering (Rayleigh) – energy of the photon is unchanged A 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

13. Metal Nanoparticle Light Scattering mixed small large 100ms integration Full Color Scattering Spectra 50 mm Darkfield Scattering Image Mixed Sample Spectra

14. Silver Nanorods

15. Surface Modification Polyethylene Glycol Dithiol (Wolfgang Eck) • Covalently bonding a polymer to the surface of the particle sterically prevents agglomeration • Thiol linked polyethylene glycol (PEG) is convenient for gold particles • Provides point of attachment of targeting molecule

17. Antibodies • Proteins produced by the immune system • Used to target foreign material such as bacteria or viruses • Convenient targeting mechanism

18. Monoclonal Antibody F19 • Developed at the Ludwig Institute for Cancer Research • Specific to FAP α (expressed in stroma) • Coupled to PEG via NHS/EDC chemistry • Purified by Size Exclusion Chromatography

19. Design Approach mAb F19 functionalized gold nanoparticles for visualization of pancreatic cancer stromal cells Y Y Y Y Y Y Y Y Y The 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

20. PEG Dithiol Synthesis Synthesizing our own allows us to control length and functionality

21. Gold Nanoparticles Broad size distribution Narrow size distribution (40 mm images)

22. SEC Column Fractions The 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.

23. TEM images of SEC fractions. Fraction 3 Fraction 8 Fraction 13

24. NIH 3T3 Cells Labeled with WGA conjugated Gold Nanoparticles

25. A 35 mm 35 mm Cancer 40x + Au-PEG - Control Cancer 40x + Au-IgG Control B

26. 35 mm 35 mm Comparison of stained vsAu-mABF19 lableled A. B. Histological Staining Labeling with F19 Conjugate

27. Cell / Ex Vivo labeling - Preliminary < 5 mm pancreatic tissue sections Negative control F-19 Nanoparticle Labeled Immunohistochemically Stained Optimization of nanoparticle geometry for optical contrast Reproducibility Lots of tissue studies Nanotoxicity (John Wise – USM)

28. Immunoelectron Microscopy Gold Scatters Electrons! Labeling with Conjugates Fixation and Embedding Viewed by TEM Labeling or Tissue Expression?

29. Conclusions: • Gold nanoconjugates have great potential in cancer diagnosis and treatment due to their optical contrast • Pegylated gold is extremely stable • MAB-F19 based conjugates show promise but more characterization is necessary.

30. Future Directions • Hard to get F19 • Problems with animal model –Different antibody? • Other contrast mechanisms -CT Contrast

31. References W 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. 1988 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):2263-72, 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, 231-5.

32. The Mason Group Gary Craig Matt King Ray Kennard Ed Allgeyer David Paul Sara Sterling Ben Freedman SushilKadka ArunaSigdel Mike Browne Adam Pongan Eric Young Jennifer Brown EbenEstell Maria Collins Collaborators William DeSisto Sam Hess David Neivandt Peter Allen Wolfgang Eck Thank You! Support: NSF, Memorial Sloan Kettering Cancer Center

33. Excessive intake of Silver colloids (Argyria) Questions?