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Nano-imaging methods for visualization of nanoparticles in vitro and in vivo: Analysis of uptake, distribution and quantitative assessment
Project
Project code: BfR-CPS-08-1322-502
Contract period: 01.05.2012
- 31.12.2016
Purpose of research: Basic research
Increasing numbers of products furnished with nanoparticles require careful analysis of possible adverse health effects in humans. By reducing particle size toward nanoscale ranges novel and partially unexpected physicochemical properties appear. Sometimes such properties are of advantage and favor, thus explaining the increasing abundance of nanoparticles in products at the market. However, this may lead to altered toxicological effects as well as to an altered uptake and distribution (uptake into cells, transport via barriers, tissue distribution, excretion). Such new features also hamper toxicological assessment though. It is not possible to predict the behavior of nanoparticles based on properties of the corresponding bulk material. A risk assessment requires data on the hazard (e.g. toxicity) as well as on the exposure (external and internal doses). In both areas we have huge data gaps. Currently reliable risk assessments are impossible. There is a strong need to develop new methods and to adopt the existing ones. One of the main challenges is to determine the internal dose of nanoparticles. Robust methods to quantify the uptake of nanoparticles in organisms, tissues or cells with high accuracy are lacking. As well we need methods to analyze the precise intracellular distribution with high resolution in space and time. Therefore we test and establish different methods suitable to study uptake and distribution of nanoparticles in vitro and in vivo, possibly even in a quantitative manner.
2017: Increasing numbers of products furnished with nanoparticles require careful analysis of possible adverse health effects in humans. Nanomaterials may show altered behavior compared to conventional materials with respect to cellular uptake, transport via barriers or distribution. Thus, the behavior of nanomaterials currently cannot be predicted based on properties or behavior of conventional materials but has to be tested experimentally in a case-by-case approach. In order to do so, there is a strong need to develop new methods and to adopt the existing ones. Therefore within this project we will test, establish and compare different methods suitable to study uptake and distribution of nanoparticles in a quantitative manner. For this purpose we will apply different methods such as flow cytometry and confocal laser scanning microscopy and in cooperation with other institutes also electon microscopy, x-ray microscopy or different ICP-MS based methods. The second part of this project analyses how nanoparticles change in biological environments where they may interact with proteins forming a so-called protein corona that can influence cellular uptake in vitro. This project shall investigate the role of the protein corona for cellular uptake of nanoparticles. The last part of this project deals with the fate of the nanoparticles after the cellular uptake. For this purpose we will develop nanoparticles with special coatings, being either very stable or which can be specifically cleaved in biological environment. In summary this project will test, develop and compare different techniques suitable to study and to quantify nanoparticle uptake. These techniques will then be applied to investigate the role of the protein corona for nanoparticle uptake and the stability of the nanoparticles in biological environment.
Within this project we have successfully applied different methods to study nanoparticle uptake in cells. Each of those methods had specific advantages but also certain limitations. We could successfully apply flow cytometry and confocal microscopy to investigate the cellular uptake of fluorescently labeled SiO2 nanoparticles. Using such approaches may, however, been hampered by the fact that the covalent linkage of a fluorescent dye may alter the nanoparticle surface and in addition over time gradually the dye might be released, which can interfere with the measurement. In order to avoid this we have used special core- shell nanoparticles, where after the attachment of the dye another silica layer was applied. This allowed us to study the cellular uptake of three different sizes of SiO2 nanoparticles in different cell models. We could detect size-dependent differences in cellular uptake. The smallest particles (15 nm) were taken up more efficiently and also faster into different cell models. We also found differences with respect to uptake mechanisms which were size-dependent but also dependent on the cell model used. The advantages of these fluorescence-based methods are that these techniques are widely available in biological laboratories, they do not require intense sample preparation and can deliver information on the level of single cells. A limitation is that absolute quantification is mostly not possible due to the fact that the labelling efficiency cannot be easily determined and may also vary between particles in one batch. In addition we have successfully applied different ICP-MS based methods to study the cellular uptake of two different silver and two different TiO2 particles. Using conventional ICP-MS it was possible to determine total amounts and by considering the number of cells one could determine average amounts per cell. By using single particle ICP-MS we could confirm the data obtained by conventional ICP-MS and furthermore could get information about particle sizes. Using Laser-Ablations ICP-MS we could investigate the uptake on single cell level and found a large heterogeneity in particle uptake between different cells in the same culture. Using the different ICP-MS approaches we could also confirm size-dependent differences in nanoparticle uptake. By comparing the amounts of particles taking up based on nanoparticle masses, clearly the bigger silver and TiO2 particles were taken up more efficiently. However when comparing amounts of particles taking up based on particle numbers, the smaller particles were taken up more efficiently. In summary ICP-MS based techniques are very useful to study uptake of metal based nanoparticles and by using variants it is possible to get information about nanoparticle sizes or information of uptake into individual cells. In addition we could apply a rather new approach based on Focused Ion Beam in combination with scanning electron microscopy (FIB-SEM) to quantify the number of silver nanoparticles in a single cell. To that end we have developed a mathematical algorithm to perform automated analysis. The result was confirmed using ICP-MS and transmission electron microscopy. Finally we could successfully investigate the influence of the protein corona for cellular uptake using flow cytometry and confocal microscopy. We could not only see clear differences in cellular uptake in presence or absence of a protein corona as also observed by others but we could identify individual proteins that might be involved mediating the uptake. In summary all objectives of this project have been reached.
Section overview
Subjects
- Food Processing
- Toxicology
