Personnel
Wolfgang Eck
The Jackson Laboratory
Development of magnetic nanoparticles with biomolecularly defined coatings
We are planning a joint project with groups from the Institute for Molecular Biophysics, the Universities of Heidelberg and Bielefeld in Germany, and two groups from the Netherlands. The main goal is the development of a novel magneto-optical nanotechnology that will allow analysis of biomolecular systems inside living cells and tissues. The technology is based on the introduction of single highly magnetic nanoparticles into cells, which can be visualized in time and space using standard microscopy and can be moved and positioned inside a cell using magnetic tweezers. These magnetic nanobeads will be biochemically functionalized by attaching them to biomacromolecules, such as specific proteins, RNA or DNA molecules, and subsequently used to analyze and manipulate specific molecular systems inside living cells. This novel nanotechnology may open new ways for the analysis of molecular processes in living cells and tissues. The emphasis of the whole project is on tracking and manipulating intra-cellular nanoparticles, however when attached to a specific probe molecule (e.g. antibodies, transcription factors), the nanoparticle may also become an intracellular sensor or a device to manipulate intracellular processes. The coatings to be developed for the nanoparticles, both to prevent non-specific adsorption and to bind specifically to proteins or DNA, may further facilitate the development of new diagnostic methods.
Specifically, magnetic nanoparticles (as prepared by the Bielefeld group) will be coated with self-assembled monolayers so they will become biocompatible, can be further biofunctionalized, and show minimal non-specific binding of proteins and nucleic acids. Core-shell nanoparticles (e.g. cobalt or iron oxide coated with gold or platinum) will be functionalized with chemically defined self-assembled monolayers using polydisperse polyethylene glycol, phosphorylcholine or short oligosaccharide thiols. The typical chain length distribution of polydisperse PEG will then allow the immobilization of proteins, DNA fragments or oligosaccharides on the particles, while full biocompatibility of the system is retained. Chemical anchoring of the PEG macromolecules to the surface will be accomplished via terminal thiol groups on gold and platinum covered particles. Initially, we will use carboxy terminated PEG thiols that have already been synthesized and characterized on planar gold surfaces.
Proteins, antibodies or oligonucleotides will be coupled to the terminal carboxy groups of these surface layers (e.g., using standard carbodiimide coupling chemistry). The average molecular weight of the PEG units can be varied between 2,000 and 10,000 in order to achieve an optimum compromise between total particle size, solution stability, biocompatibility and selectivity of the attached signal molecules. As an alternative biocompatible coating system, oligosaccharide terminated thiols, such as those described by Mrksich and coworkers can be used to cover the core-shell nanoparticles.
After preparation, the size-distribution and shape of the particles will be determined by scanning electron microscopy. By measuring the light absorption of the dispersion, the stability of the particles in salt solutions, protein containing medium or serum will be examined. The mobility and stability of the particles and their dispersions in magnetic fields will be determined using methods available from the different partners. Most likely, the stability and mobility can be tuned using different molecular weights of the PEG coatings.
Such chemically highly defined biofunctionalized particles are expected to be directable by magnetic tweezers within a cell in a very precise way. In collaboration with the partner groups, their interaction with complementary biological entities will be examined in vitro and in vivo.
Free-standing phospholipid membranes on nanostructured artificial cytoskeletons - Two-dimensional models for cell membranes
We wish to develop the preparation of novel phospholipid bilayers that are anchored on nanostructured surfaces and may serve as realistic two-dimensional models of natural cell membranes. Since natural cell membranes are anchored on a cytoskeleton on regularly spaced protein arrays with lateral distances of several 10 to more than 100 nm, we wish to mimic this situation using lithographic or self-assembling methods. This way, we hope to generate free-standing membranes that provide ample free submembrane space capable of accommodating the intracellular portions of membrane proteins and thus will not alter the natural protein conformation or lateral mobility within the membrane.
Such nanostructured artificial cytoskeletons can be prepared either by electron beam lithography or by self-assembling methods, providing e.g. chemically nanostructured surfaces or regularly arranged nanopillars as developed by Spatz and co-workers. These regularly spaced nanoarrays may be chemically converted into an artificial cytoskeleton e.g. by functionalization with avidin, thus providing avidin nanopatterns to which commercially available biotinylated phospholipids can be coupled.
Initial questions to be addressed in the development of this idea will include the optimization of array spacings and geometries and the determination of the structure of the adsorbed lipids, i.e. whether real bilayers are present or lipid multilayers or vesicles instead. Physical methods to be utilized may include ellipsometry, sum frequency generation, neutron scattering or FTIR spectroscopy.
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