1. Ferric Iron Nanoparticles Formation Mediated By Negatively Charged Polypeptides Vamsi Krishna Aluru, Dr Robin S. Hissam Department of Chemical Engineering, West Virginia University,Morgantown, WV November 9, 2010 AICHE 2010 Annual Meeting

2.

Background

Objective

Research

Results

Conclusions

Future Work

Acknowledgement

Outline AICHE 2010 Annual Meeting 3. Background

Biomineralization

• • Examples:Silicates in algae, carbonates in invertebrates,

• • magnetite in magnetotactic bacteria

Magnetotactic Bacteria

Transmission Electron photograph of magnetotactic bacterium and bacterial magnetic particles A : Magnetospirillum magneticum AMB-1 * B : Desulfovibrio magneticum RS-1** * Matsunaga T et al. Appl Microbiol Biotechnol, 1991 ** SakaguchiT Matsunaga T et al. Nature, 1993 AICHE 2010 Annual Meeting 4. Magnetic Particles

Two types

• Iron oxides (Fe 3 O 4 ) and iron sulfides (Fe 3 S 4 , FeS 2 )

All magnetic particles are of size 35-120 nm

Morphology of crystals

• cuboidal,

• elongated prismatic and

• arrowhead-shaped

Stable single magnetic domains

(SMD)

http://scienceblogs.com/afarensis/2006/01/24/more_on_magnetic_bacteria/ AICHE 2010 Annual Meeting 5. Applications of Magnetic particles

Magnetic drug delivery system

DNA extraction

Magnetic Resonance Imaging (MRI)

*Hypothetical magnetic drug delivery system * Pankhurst Q A et al. J.Phys. 2003 AICHE 2010 Annual Meeting Magnetic Nanoparticles Tumor DDS Diagnosis Therapy Hyperthermia MRI MI sensor 6. Synthesis of Magnetic Particles

Magnetosomevesicle formation

• • • Invagination of cytoplasmic membrane

Uptake of iron into vesicle

Biomineralization

Fe +3 Fe +2 +1 vesicle AICHE 2010 Annual Meeting 7. Magnetosome

Magnetosome

• • Cell organelle with magnetite crystal and an outer cover of lipid bilayer membrane

Membrane Composition

• • Phospholipids, fatty acids and proteins

Proteins are specific to membrane and are responsible for crystal growth

Arakaki et al. characterized a number of proteins inMagnetospirillum magneticumstrain AMB-1

AICHE 2010 Annual Meeting 8. Biomineralization Protein-Mms6

All proteins isolated had hydrophobic N-terminal region and a hydrophilic C-terminal region

Magnetite nanoparticles with similar morphology to bacterial magnetite particles were synthesized in presence of recombinant Mms6 expressed inE.coli

* Electron micrographs of magnetic particles synthesized in the presence (A) and absence (B) of Mms6. *Arakaki A et al. J. Biol. Chem 2003AICHE 2010 Annual Meeting 9. Mms6 Protein

Hydrophilic C-terminal region of Mms6 contains amino acid residues with hydroxyl or carboxyl groups

Arakaki et al. proposed that Mms6 acts as a template for magnetic nanoparticle synthesis

AICHE 2010 Annual Meeting G A V Y A Y M K S R D I E S A Q S D E E V E L R D 10. Advantages of Biomimetics

Chemical processes

• High temperature, pressure and pH

• Low yield , difficult to reproduce

• Often produce toxic byproducts

Biomimetics is a natural way to produce nanoparticles of similar morphology to those produced in magnetotactic bacteria

AICHE 2010 Annual Meeting 11. Objective

Central hypothesis

• • Effect of hydroxyl and carboxyl groups on size and shape of nucleated particles

Aim

To design peptides with different functional groups

To inspect the morphology of crystals formed with different peptides

AICHE 2010 Annual Meeting 12. Design of Peptides AICHE 2010 Annual Meeting Name Peptide VA1 M G A V Y A Y G K S R S I Y S A Q S Y S S V S L R Y (OH) VA2 M G A V D A D G K E R D I D E A Q E D E E V D L R D (COOH) VA3 M G S E D A Y G S Y D E S Y D E Q S E D Y E S Y Y S (Combination) VAMms6 M G A V Y A Y G K S R D I E S A Q S D E E V E L R D 13.

Background

Objective

Research

Results

Conclusions

Future Work

Acknowledgement

AICHE 2010 Annual Meeting 14. SDS-PAGE Protein purification profile of VA-Mms6, VA1 and VA2, 12.5 % separating gel, ran @100V: Lane 1) Protein ladder, Lane 2) VA-Mms6, Lane 3) VA1 and Lane 4) VA2 AICHE 2010 Annual Meeting 37 kD 25 kD 20 kD 15 kD 10 kD VA-Mms6 VA1 VA2 1234 15. Western Blot Western Blot Stained with anti-His antibody: Lane1) purified His-tagged VA-Mms6;Lane 2) purified His-tagged VA1; Lane 3) purified His-tagged VA2 Courtesy: Dr. Yogesh Kulkarni AICHE 2010 Annual Meeting 16. Experiment

Co-precipitation reactions were carried out using all peptides

100l of 0.6M FeCl 3and 0.3M FeCl 2were added to 10l of peptide solution ( 5mg/ml)

0.1N NaOH was titrated against above reaction mixture under constant nitrogen flow

The color of solution turned from pale yellow to black

All solutions were degassed with argon prior to reaction

Particles were allowed to grow for 7 days and concentrated at the bottom using neodymium-boron magnet.

AICHE 2010 Annual Meeting 17. Scanning Electron Microscopy (SEM) (a) SEM image of iron particles synthesized in the presence of VA-Mms6 (b) EDS of iron particles synthesized in the presence of VA-Mms6 AICHE 2010 Annual Meeting (c) SEM image of iron particles synthesized in the presence of VA2(d) SEM image of iron particles synthesized in the presence of VA3(c) (d) 18. Transmission Electron Microscopy (TEM) TEM images of magnetite particles synthesized in presence of : (a) VA2 (b) Mms6 VA2 *Mms6 Courtesy:Dr. Chaoying Ni,University of Delaware *Arakaki A et al. J. Biol. Chem 2003AICHE 2010 Annual Meeting Selected Area Electron Diffraction (SAED) patterns of iron oxide particles synthesized in the presence of VA2 (a) (b) 19. Atomic Force Microscopy (AFM) (a) (b) (c) AFM image of magnetite particles synthesized in the presence of (a) VA-Mms6, (b) VA2, (c) VA3 Courtesy: Srikanth Raghavan, WVU AICHE 2010 Annual Meeting 20. X-ray Photoelectron Spectroscopy (XPS) XPS spectra of magnetite particles synthesized in the presence of VA2, VA3, VA-Mms6 and XPS spectra of standard magnetite particles711 eV 724.4 eV VA-Mms6 Courtesy: Srikanth Raghavan, WVU AICHE 2010 Annual Meeting 21. DLS AICHE 2010 Annual Meeting Size distribution of magnetiteparticles synthesized in the presence of (a) VAMms6, (b) VA2, and (c) VA3 Size (d,nm) Size (d,nm) Size (d,nm) Number (%) Number (%) Number (%) a) b) c) Peak Diameter VAMms6 88 nm VA2 62 nm VA3 41 nm 22. AICHE 2010 Annual Meeting 23. Magnetic Force Microscopy (MFM) MFM image of magnetite particles synthesized in presence of VA2 (a) topography (b) phase (a) (b) Courtesy: Srikanth Raghavan, WVU AICHE 2010 Annual Meeting Individual particles of size range 40 nm 24. Conclusions

Magnetite nanoparticles were synthesized with the aid of VA-Mms6, VA2 and VA3

Discrete particles of size range 60 nm were observed in the presence ofVA2

Magnetite particles synthesized in the presence ofVA2 showed stable magnetic domain

Carboxyl group peptide VA2 showed a variation in size and morphology of particles in comparison to VA-Mms6 and VA3

AICHE 2010 Annual Meeting 25. Future Work

In the near future characterization of particles synthesized in the presence of PEG-VA2 and PEG-VA3 should be carried out

Film coating of the iron particles with a biocompatible polymer like PEG will allow the particles to be used as drug delivery agents.

Functionalized Magnetite nanoparticles can even be used as contrast agents in MRI

AICHE 2010 Annual Meeting 26. Preliminary Work

Iron particles were synthesized in the presence of polyethylene glycol (PEG) coupled to the peptides VA2 and VA3

(a) (b) SEM image of magnetite particles synthesized in presence of (a) PEG-VA2 (b) PEG-VA3 AICHE 2010 Annual Meeting 27. Acknowledgement

Dr. Robin Hissam

The Hissam Group

• • Avram Siegel, Jaclyn Kokx, Logan May, and Andrew Graves

Dr. Charter D. Stinespring and Srikanth Raghavan

Dr. Rajesh Naik (AFRL)

Liviu Magean and Adrienne McGraw

Dr. Chaoying Ni, Dr. Thomas Epps, and Julie N. L. Albert (University of Delaware)

WVU Research Corporation for funding

AICHE 2010 Annual Meeting 28. Thank You AICHE 2010 Annual Meeting