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?