PEGylated Gold Nanoparticles Conjugated to Monoclonal F19 Antibodies as Targeted
Labeling Agents for Human Pancreatic Carcinoma Tissue
February 26, 2009
Gary CraigDepartment of Chemical and Biological Engineering
University of Maine
Sections of Talk
1. Pancreatic Cancer 2. Noble Metal Nanoparticles 3. Functionalization and Bio-Linking
4. Tissue Studies 5. Conclusions 6. Future Directions
Application to Pancreatic Cancer Detection
Early diagnosis – whole animal imaging (x-ray tomography)
Help the surgeon identify cancer margins during surgery.
Pancreatic Cancer produceslow density tumor masses:
Not visible early by screeningmethods (CT scanning)
During surgical removal, the tumor margins are difficult tofind.
Histology of Pancreatic Adenocarcinoma
(Standard Diagnostic Method)
Healthy Pancreatic Tissue
Standard Histology Stain Labeled for Cancer Stroma
Histology of Pancreatic Adenocarcinoma
Pancreatic Cancer Tissue
Standard Histology Stain Cancer Stroma Labeled
Interconnected Themes of Interest in our Lab
Observation of (Bio)-Chemical processes at the single-molecule level in heterogeneous systems
Nanoprobe Photophysics – Understanding Chemical and Physical characteristics
Developing nanoprobes for imaging at the single molecule level (Engineering and synthesis)
Development of imaging tools for single molecule detection
Sample Driven Design Constraints:Question (material or animal system)GeometryChemical/Physical EnvironmentTime scale
Measurement
(1)
(2)
(3) (Tissue)
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
•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
Stirring
•Size depends on the molar ratio of metal ion and reducing agent
timeAu0
Metallic Nanoparticles – Batch Synthesis
•Rod shaped particles formed via surfactant templating (CTAB, TOAB)
Au3+
OstwaldRipening
Reducing Stable Colloid
Metallic Nanoparticles
Size and geometry tunable (2.5 – 65 nm)
Very expensive to buy (spheres), relatively cheap and easy to make
Gold
(spheres)
Gold
(Rods)
Photo-physics??
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
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
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
Part I. Simple Light Scattering
26
4
1
2Scattered
r mI
mλ−
∝+
Metal Nanoparticle Light Scattering
550 600 650 700 750 800
Wavelength (nm)
Scattering Intensity
small
large
mixed
50 mm
Mixed Sample Spectra Darkfield Scattering Image
100ms integrationFull ColorScattering Spectra
Silver Nanorods
Surface Modification 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
(CH2)9 (OCH2CH2)n
SHHS
OCH2COOH
Polyethylene Glycol Dithiol(Wolfgang Eck)
QuickTime™ and aYUV420 codec decompressor
are needed to see this picture.
Antibodies
Proteins produced by the immune system
Used to target foreign material such as bacteria or viruses
Convenient targeting mechanism
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
YY
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
Design ApproachmAb F19 functionalized gold nanoparticles for visualization
of pancreatic cancer stromal cells
YYYY
YYYY
YY
YYYY
YY
PEG Dithiol Synthesis
(CH2)9 (OCH2CH2)n OCH2COOH
(CH2)9 OHO
NaH / DMF / 60°C
(CH2)9 (OCH2CH2)n O-Na+
+
1. ClCH2-COONa / 60°C
2. HCl
I
II
(CH2)9 (OCH2CH2)n
SHHS
OCH2COOH
(CH2)9 (OCH2CH2)n
BrBr
OCH2COOH
Br2
1. NaSH / H2O
2. acetic acid
III
IV
Synthesizing our own allows us to control length and functionality
Gold Nanoparticles
Narrow size distribution Broad size distribution
(40 mm images)
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.
SEC Column Fractions
Fraction 3 Fraction 8 Fraction 13
TEM images of SEC fractions.
NIH 3T3 Cells Labeled with WGA NIH 3T3 Cells Labeled with WGA conjugated Gold Nanoparticlesconjugated Gold Nanoparticles
Cancer 40x + Au-PEG - Control Cancer 40x + Au-IgG Control
35 mm35 mm
A B
Comparison of stained vs Au-mABF19 lableled
35 mm35 mm
Histological Staining Labeling with F19 Conjugate
A. B.
Cell / Ex Vivo labeling - Preliminary
< 5 mm pancreatic tissue sections
Optimization of nanoparticle geometry for optical contrast
Reproducibility
Lots of tissue studies
Nanotoxicity (John Wise – USM)
ImmunohistochemicallyStained
F-19 NanoparticleLabeled
Negative control
Immunoelectron Microscopy
Gold Scatters Electrons! Labeling with Conjugates Fixation and Embedding Viewed by TEM Labeling or Tissue Expression?
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.
Future Directions
Hard to get F19
Problems with animal model –Different antibody?
Other contrast mechanisms-CT Contrast
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.
The Mason Group
CollaboratorsWilliam DeSistoSam HessDavid NeivandtPeter AllenWolfgang Eck
Gary CraigMatt KingRay KennardEd AllgeyerDavid PaulSara Sterling
Ben FreedmanSushil KadkaAruna SigdelMike BrowneAdam PonganEric YoungJennifer BrownEben EstellMaria Collins
Thank You!
Support: NSF, Memorial Sloan Kettering Cancer Center
Excessive intake of Silver colloids (Argyria)
Questions?
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