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Nanoparticles: researchers to map protein corona

The way nanoparticles behave in the human body not only depends on their chemical structure. Of greater importance is the way they interact with biological molecules. Professor Gerd Ulrich Nienhaus at the Karlsruhe Institute of Technology (KIT) has developed new methods that enable the quantitative measurement of these dynamic processes.

Proteins (cyan) enclosing a nanoparticle (green) which, like the free protein, can bind to the cell surface, e.g. to receptors (blue) © CFN

When nanoparticles enter the blood, they immediately become coated with a thin layer of biomolecules. This biological surface coating, which is referred to as protein corona, determines to a great extent whether the protein is excreted or whether it is able to enter a body cell. "Nanoparticles that enter the body by mistake, need to be removed as quickly as possible. However, in cases where nanoparticles are destined for therapeutic use, they need to be taken up by certain types of cells. It is therefore important to understand how molecules in the body are able to bind to nanoparticles since a nanoparticle interacts with the cell surface by way of this monolayer," explains Nienhaus who has recently moved from the University of Ulm to the Centre for Functional Nanostructures at the KIT. Nienhaus' investigational methods, which were recently published in the renowned scientific journal Nature Nanotechnology, enable him to work on these issues experimentally.

The biophysicist chose serum albumin, which is a key blood protein, as model protein. The diameter of a nanoparticle increases when the protein binds to its surface. Nanoparticles move around constantly in an aqueous solution and this diffusion movement decreases as the nanoparticles increase in size. In order to determine the thickness of the protein layer on a nanoparticle, Nienhaus and his team are currently measuring the time a nanoparticle takes to move through a small volume.

The nanoparticles send out fluorescence light when irradiated with light. This enables them to be seen by the researchers despite their tiny diameter of just six to eight nanometres (1 nanometre = 1 millionth millimetre). If a particle passes through an extremely small fluid volume in the examination chamber of a specifically developed microscope, it is hit by a laser beam and emits light for a fraction of a second. The length of the light flash can be measured precisely. If the flash is short, the particle is moving rapidly, if it is long, the particle is moving slowly, which suggests that it has a larger diameter. "Since we know the size of an albumin molecule, specific formulas used in physics can be used to calculate the overall size of the particle. We found that the protein layer on a nanoparticle consists of only one molecule layer," said Nienhaus summarising their results.

But how long does it take to create this coating, and how stable is it? In order to find answers to these questions, the researchers labelled the proteins with a dye that weakens the fluorescence of the nanoparticle. When the protein molecules treated in this way bind to a particle, the particle's light intensity diminishes. The measurement data show that a serum albumin molecule adheres to the particle surface for about 100 seconds on average before it detaches and is replaced by another molecule.


Nienhaus and his team now plan to investigate other combinations of different biomolecules and nanoparticles. They will also carry out cell culture experiments in order to find out how cells react to the enveloped nanoparticles. The method developed by the Karlsruhe researcher opens up new possibilities for measurement, which are also important for the assessment of the potential risk of nanoparticles for human health - an issue that is raised in a contribution on the team's findings in the "News and Views" section of the journal Nature Nanotechnology.

Fluorescence correlation spectroscopy:

Fluorescence correlation spectroscopy (FCS) can be used to investigate the dynamic properties of particles and of individual molecules in solution. Photons, which are emitted by fluorescent objects upon excitation with laser light, are registered in a confocal microscope as a function of time. Since the laser beam is extremely focused, the sample volume investigated is only about one femtolitre (1 femtolitre = 1 quadrillionth litre). The fluorescence intensity measured (number of photons per time interval) fluctuates because individual, labelled molecules diffuse in and out of the sample volume as a result of Brownian motion or because chemical or physical molecule alterations alter the emission of light. The statistical analysis of the fluctuations enables the precise determination of diffusion constants and rest times.

Literature:

A quantitative fluorescence study of protein monolayer formation on colloidal nanoparticles. Carlheinz Röcker, Matthias Pötzl, Feng Zhang, Wolfgang J. Parak and G. Ulrich Nienhaus. Nature Nanotechnology 4, 577 (2009).

What does the cell see? Iseult Lynch, Anna Salvati and Kenneth A. Dawson. (News & Views) Nature Nanotechnology 4, 546 (2009).

 

Further information:
DFG - Centre for Functional Nanostructures (CFN)
Dr. Gerd König
Wolfgang-Gaede-Str. 1a
76131 Karlsruhe
Tel.: +49 721 608-3409
Fax: +49 721 608-8496
E-mail: gerd.koenig(at)cfn.uni-karlsruhe de

 

Website address: https://www.gesundheitsindustrie-bw.de/en/article/press-release/nanoparticles-researchers-to-map-protein-corona