Magnetic nano- and microparticles in bioscience

and biotechnology

Ivo Safarik and Mirka Safarikova

Ceske Budejovice, Czech Republic

Type of magnetic nano- and microparticles

Multi domain, single domain or superparamagnetic

• Magnetite (Fe3O4)

• Ferrites (MeO . Fe2O3; Me = Ni, Co, Mg, Zn, Mn)

• Maghemite (-Fe2O3)

• Greigite (Fe3S4)

• Iron, nickel

Magnetic nano- and microparticles

Inorganic material, but:

• Present in biological systems

• Usable for a variety of bio-applications

Important dates

• 1962 - Lowenstam (biochemically-precipitated magnetite as a capping material in the radula (tongue plate) teeth of chitons (marine mollusks of the class Polyplacophora)

• 1975 - Blakemore (magnetotactic bacteria)

Magnetic material in teeth of chitons

• hardening the tooth caps (enabling the chitons to extract and eat endolithic algae from within the outer few millimeters of rock substrates)

Magnetotactic bacteria

• Various morphological types exist

• Gram-negative prokaryotes

• Magnetic nanoparticles present in magnetosomes

• Found in sediments in diverse aquatic environments

Magnetotactic bacteria

Provided by Dr. D. Schüler, Germany

Magnetosomes

• Composed from magnetite (Fe3O4) or greigite (Fe3S4)

• Ca 50 nm in diameter• Covered with a lipidic membrane• Important for navigation of the bacteria into

oxic/anoxic layer • Biocompatible character (phospholipid bilayer)• Possible applications in biosciences

Magnetosomes

Provided by Dr. D. Schüler, Germany

Magnetosomes

Provided by Dr. D. Schüler, Germany

Biogenic magnetic nanoparticles

• Magnetotactic bacteria

• Magnetotactic algae

• Protozoa

• Insect

• Fish

• Amphibians

• Reptiles

• Birds

• Mammals

Animal magnetite

• Magnetosome chains from the frontal tissues of chinook salmon

S. Mann et al.: J. Exp. Biol. 140 (1988) 35-49.

Importance of animal magnetite

• Most probably involved in magnetoreception (what is the current position ???, which direction to move ???) interaction of magnetite nanoparticles (chains of magnetic nanoparticles ?) with the sensing cells

Non-physiological presence of magnetic nanoparticles

• Found in human brain in case of neurodegenerative diseases

• Disruption of normal iron homeostasis in the brain

• Early detection of neurodegenerative diseases using MRI ??

Magnetic nanoparticles in meteorites from Mars

• Meteorite magnetic nanoparticles resemble magnetosomes from Earth magnetotactic bacteria

• Hypothesis: Was there a transfer of living systems from Mars to Earth???

Let’s return back to Earth !!

Magnetic nano- and microstructured

materials for bioapplications • Useful for many applications in

biosciences, biotechnologies and environmental technologies

• Enable to label diamagnetic structures • Enable separation or detection of

magnetically labeled structures• Both separation and non-separation

applications are important

Which nano- and microstructured magnetic materials?

Three important bio-• Biocompatible (in the broadest sense of this

word)

• Biodegradable

• Biomimetic

Biocompatible materials

Biocompatibility in the broadest sense of the word• Minimal toxicity • Stability of nanoparticles in body fluids• Possibility for immobilization of biologically active

compounds, cell organelles, cells (activated nanoparticles)• Reproducible interaction with biosystems• Enable labeling of biostructures (for magnetoassays)• Biopolymer based particles, PEGylated structures,

magnetoliposomes, biopolymer-based FFs, self-assembled magnetic structures

Biodegradable materials

• Important from the point of view of possible clinical applications (e.g., drug targeting)

• Nanoparticles formed by biodegradable (bio)polymer

• Magnetoliposomes and related structures

• Starch-based magnetic fluids

Biomimetic materials

• Analogues of bacterial magnetosomes

• Ferritin x magnetoferritin

Important data

• 1960s – Pappell (first ferrofluids, 1000 hours of magnetite milling)

• 1966 - Magnetically controlled thrombosis of intracranial aneurysms

• 1970s – increase of interest in biomagnetic research and technology

• 1980 – Massart (chemical synthesis of aqueous ferrofluid without detergents)

• 1980s – commercially available magnetic particles

Magnetic fluids – examples of magnetic nanostructured materials

• ferrofluid can be influenced by the presence of external magnetic field

Synthetic biocompatible magnetic nanoparticles

and complexes containing them • Biocompatible magnetic

fluids

• Magnetoliposomes

• Magnetic nano- and microparticles

• Magnetoferritin

Magnetic microparticles

Dynabeads• Homogeneous size

distribution

• Simple recovery with small permanent magnets

• Magnetic nanoparticles present inside the beads

• Available in plain, activated and bioactive form

Examples of of magnetic nano- and microparticles applications

• From molecular biology to environmental technologies

• Manipulation of microliters as well as million of liters

• Manipulation in suspension systems

• Both separation and non-separation techniques are important

Immobilization and modification of biologically active compoundsPossibility of simple removal of the immobilized

molecules or cells from the system • Immobilization of enzymes, antibodies, lectins,

oligonucleotides, affinity ligands• Modification of proteins via magnetic PEG• Immobilization in magnetoliposomes• Magnetosomes from genetically engineered

magnetotactic bacteria carrying target proteins

Isolation of biologically active compounds

• Direct separation of targets from crude samples containing solids• Affinity magnetic beads or magnetic two-phase systems• Preconcentration of target molecules from large volumes of crude

materials

• Poly A+ mRNA, DNA, oligonucleotides • Enzymes• Lectins• DNA/RNA binding proteins• Antibodies, antigens • Xenobiotics (both organic and inorganic)

High gradient magnetic separators

Test tube magnetic separators

Automated systems

KingFisher mL

Thermo, USA

KingFisher 96Thermo, USA

Te-MagSTecan, Japan

Determination and detection of biologically

active compounds and xenobiotics • Immunomagnetic assays (magnetic particles used

for separation of target analyte)• Magnetoimmunoassays (magnetic particles serve

as detectable labels)• Magnetic substrates• Magnetic solid-phase extraction• New principles for the detection and quantitation

of both magnetic labels and biological structures tested (giant magnetoresistance, magnetoelasticity, magnetic atomic force microscopy…)

Automated systems for immunoassays

• Combination of specific magnetic separation and sensitive detection (enzyme, radioactive, chemiluminescent or fluorescent)

• Different assay formats can be used (sandwich x competitive immunoassays)

• Generic solid phases can be used (streptavidin, secondary antibody, protein A …)

Beckman Coulter's Access Immunoassay System

Magnetoimmunoassays

• Magnetic nanoparticles serve as a label

• Possibility to replace enzymes, radioisotopes …

• Magnetic Permeability Reader (Euris, Sweden, top; Magnetic Assay Reader (Quantum design, USA, bottom)

Magnetic solid-phase extraction

Detection of crystal violet in water

• Magnetite with immobilized phthalocyanine dye used as a selective adsorbent

• Curve B – 0.5 μg of dye in 1000 ml of water

• Sensitivity 0.5 ppb achieved

Safarik,I.,  Safarikova, M.: Detection of low concentrations of malachite green and crystal violet in water. Water Res. 36 (2002) 196

Cells

• Immunomagnetic separation of target cells and cell organelles

• Detection and separation of cancer cells• Isolation of stem cells• Separation of microbial pathogens • Study of cell function • Transfection of cells • Magnetic twisting cytometry • Magnetic labeling of cells• MRI tracking of transplanted cells (magnet. modified)

Labeling of T-lymphocytes with magnetic nano- and microparticles

E. coli O157 bound to Dynabeads

Principle of IMS

Large-scale immunomagnetic separation of cells

• Several magnetic separation devices commercially available

• CliniMACS employs magnetic nanoparticles for cells labeling and HGMS system for separation

Magnetotactic bacteria

• Biotechnology production of native or genetically engineered magnetosomes

• Non-destructive domain analysis

• Radionuclide recovery

Drug and radionuclide targeting

• Accumulation of active compounds in the target organ or tumor

• Ferrofluids, magnetoliposomes, ferrofluid modified erythrocytes, magnetic polymer composites

Magnetic drug targeting

• MTC - composite of elemental iron and activated carbon with anticancer drug adsorbed onto it (FeRx Inc., San Diego, CA, USA)

Magnetic Field

Body surface

Magnetic fluid hyperthermia

• Cancer treatment

• Heating of tissues labeled with magnetic particles to 42 - 46 ºC after exposure to alternating magnetic field

• Magnetic fluids, magnetoliposomes, magnetic single- or multi-domain particles

• Combination with drug targeting

Sketch of the first prototype MFH therapy system (MFH Hyperthermiesysteme GmbH, Berlin, Germany) – Prof. Jordan

Contrast increasing during MRI

• Superparamagnetic contrast agents

• Biocompatible ferrofluids, magnetoliposomes

• Antibody-conjugated magnetite nanoparticles

MRI of bone marrow

• PEGylated magnetoliposomes as a bone marrow-seeking MR contrast agent

Potential medical applications

• Treatment of AIDS

• Endocytosis of ferrofluids by cancer cells

• Brain tumor sensing

• Artificial sphincter muscle

• Magnetic polymers (artificial muscle?)

Environmental chemistry and technology

• Waste water and potable water purification using magnetite microparticles (Sirofloc process – Australia)

• Magnetically modified microbial cells for xenobiotics removal

• Magnetic solid-phase extraction for the preconcentration of the target analytes

• Magnetically modified enzymes for the detection of their inhibitors (heavy metals, pesticides etc…)

Scheme of Sirofloc Process

Inhibition of magnetically modified trypsin 2D Graph 1

-log [inhibitor] (mol.l-1)

1 2 3 4 5 6 7 8Try

psin

act

ivity

inhi

bitio

n (%

)

0

20

40

60

80

100

- log [inhibitor] (mg ml-1)

-1.0 -0.5 0.0 0.5 1.0 1.5 2.0

Try

psin

act

ivity

inhi

bitio

n (%

)

10

20

30

40

50

60

70

Dependence of the inhibition of the trypsin activity on the concentration of Ag+ (●) and Pb2+ (○).

Dependence of the inhibition of the trypsin activity on the concentration of thionin (), 4-aminobenzamidin (), safranin (○) and bacitracin (▼).

Adsorption of magnetic nanoparticles on yeast cells

• Interaction of Saccharomyces cerevisiae cells (bakers yeast, brewers yeast) with water-based ferrofluids

• Magnetically modified yeast cells can be used as an adsorbent for the separation of biologically active compounds and xenobiotics (both organic and inorganic)

• Adsorption of organic dyes is presented

Dye Q (mg ml-1)

Q’ (mg g-1)

Acridine orange

13.1 82.8

Aniline blue 68.1 430.2

Crystal violet 13.6 85.9

Malachite green

3.1 19.6

Safranine O 14.3 90.3

Future trends

• Biotechnology production of magnetic non-modified and modified nanoparticles

• Medical applications• Magnetic nanoparticles as labels• Magnetic nanotubes • Biotechnology • Integration of magnetic nano/microparticles with

other progressive technologies (microfluidics, chips, MEMS …..)

Biomagnetic research and technology

• Multidisciplinary research area leading to new results

• Both basic research and important applications

• Creativity and fantasy is needed

• Biomagnetic research and technology is an emerging scientific discipline!

Review papers

• Safarik, I., Safarikova, M.: Magnetic nanoparticles and biosciences. Monatsh. Chem. 133 (2002) 737-759

• Safarikova,M., Safarik,I.: The application of magnetic techniques in biosciences. Magn. Electr. Sep. 10 (2001) 223-252

• Safarik,I., Safarikova,M.: Use of magnetic techniques for the isolation of cells.  J. Chromatogr. B 722 (1999) 33-53

Available on www page:

http://www.uek.cas.cz/people/safarik

• The WWW page includes also other biomagnetic information

A new on-line journal

• Journal WWW page: www.biomagres.com

• Published by BioMed Central (www.biomedcentral.com)

Edited by Ivo Safarik and Mirka Safarikova

Thank you for your attention!