Magnetic nano- and microparticles in bioscience and biotechnology

1. Magnetic nano- and microparticles in bioscience and biotechnology Ivo Safarik and Mirka Safarikova Ceske Budejovice, Czech Republic

2. 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

3. Magnetic nano- and microparticles Inorganic material, but: • Present in biological systems • Usable for a variety of bio-applications

4. 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)

5. 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)

6. Magnetotactic bacteria • Various morphological types exist • Gram-negative prokaryotes • Magnetic nanoparticles present in magnetosomes • Found in sediments in diverse aquatic environments

7. Magnetotacticbacteria Provided by Dr. D. Schüler, Germany

8. 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

9. Magnetosomes Provided by Dr. D. Schüler, Germany

10. Magnetosomes Provided by Dr. D. Schüler, Germany

11. Biogenic magnetic nanoparticles • Magnetotactic bacteria • Magnetotactic algae • Protozoa • Insect • Fish • Amphibians • Reptiles • Birds • Mammals

12. Animal magnetite • Magnetosome chains from the frontal tissues of chinook salmon S. Mann et al.:J. Exp. Biol.140(1988) 35-49.

13. 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

14. 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 ??

15. 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???

16. Let’s return back to Earth !!

17. 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

18. Which nano- and microstructured magnetic materials? Three important bio- • Biocompatible (in the broadest sense of this word) • Biodegradable • Biomimetic

19. 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

20. 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

21. Biomimetic materials • Analogues of bacterial magnetosomes • Ferritin x magnetoferritin

22. 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

23. Magnetic fluids – examples of magnetic nanostructured materials • ferrofluid can be influenced by the presence of external magnetic field

24. Synthetic biocompatible magnetic nanoparticles and complexes containing them • Biocompatible magnetic fluids • Magnetoliposomes • Magnetic nano- and microparticles • Magnetoferritin

25. 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

26. 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

27. Immobilization and modification of biologically active compounds Possibility 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

28. 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)

29. High gradient magnetic separators

30. Test tube magnetic separators

31. Automated systems KingFisher 96 Thermo, USA KingFisher mL Thermo, USA Te-MagS Tecan, Japan

32. 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…)

33. 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

34. 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)

35. 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 lowconcentrations of malachite green and crystal violet in water. Water Res. 36 (2002) 196

36. 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)

37. Labeling of T-lymphocytes with magnetic nano- and microparticles

38. E. coli O157 bound to Dynabeads

39. Principle of IMS

40. Large-scale immunomagnetic separation of cells • Several magnetic separation devices commercially available • CliniMACS employs magnetic nanoparticles for cells labeling and HGMS system for separation

41. Magnetotactic bacteria • Biotechnology production of native or genetically engineered magnetosomes • Non-destructive domain analysis • Radionuclide recovery

42. Drug and radionuclide targeting • Accumulation of active compounds in the target organ or tumor • Ferrofluids, magnetoliposomes, ferrofluid modified erythrocytes, magnetic polymer composites

43. Magnetic drug targeting Magnetic Field Body surface • MTC - composite of elemental iron and activated carbon with anticancer drugadsorbed onto it (FeRx Inc., San Diego, CA, USA)

44. Magnetic fluid hyperthermia • Cancer treatment • Heating of tissues labeled with magnetic particles to 42 - 46 ºCafter exposure to alternating magnetic field • Magnetic fluids, magnetoliposomes, magnetic single- or multi-domain particles • Combination with drug targeting

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

46. Contrast increasing during MRI • Superparamagnetic contrast agents • Biocompatible ferrofluids, magnetoliposomes • Antibody-conjugated magnetite nanoparticles

47. MRI of bone marrow • PEGylated magnetoliposomes as a bone marrow-seeking MR contrast agent

48. Potential medical applications • Treatment of AIDS • Endocytosis of ferrofluids by cancer cells • Brain tumor sensing • Artificial sphincter muscle • Magnetic polymers (artificial muscle?)

49. 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…)

50. Scheme of Sirofloc Process