Project

Nanosilberpartikel

Nanosilver particles – Mechanisms of action and investigation of their possible interaction with tissues, cells and molecules. Definition of their relevant potential for intolerance

The objective of the project Nanosilver-Particles was to develop methods for the production of silver nanoparticles (AgNP) that can be safely introduced into a product whilst maintaining the intended. From different products consisting both of permanently embedded silver nanoparticles and of releasing, resorbable systems, detailed knowledge of the respective nanoparticles and nanomaterials was to be gained to exclude potential risks of application.

Different nanosilver-containing materials were developed to serve as model systems for three product types with silver nanoparticles:

1. Organic base: Rapid short-term release of silver from e.g. collagen films
2. Metallic base: Intermediate implants for osteosynthesis e.g. titanium screws or titanium plates
3. Synthetic base: Permanent implants e.g. bone cement

Materials with different contents of silver nanoparticles were investigated to find the effective dose that enables a sufficient release and effectiveness of silver while having no toxic effect on the analysed cells. For that purpose, release profiles were analysed and the toxic effects on different cell types of the organism were determined. In addition to silver nanoparticles, conventional completely soluble silver salts were tested for comparison. Besides, the products’ antimicrobial efficiency was determined after simulated use to determine the possible duration of effect. Compatibility with the organism as well as a possible accumulation of the respective silver nanoparticles in organs were studied in limited animal experiments. No such accumulation was observed in the implants used.

The obtained results enable a better and more realistic assessment of the risk potential of silver nanoparticles and nanosilver-containing materials and allow safe assessment of the hazards when using these in medical products. According to the results, antimicrobially active nanosilver-containing medical products present a low risk to consumers while reducing the risk of implant-associated infections.

In the developed products, the silver nanoparticles show:

• a good bactericidal effect
• a good efficacy already at low dosages
• a good incorporation in the tested materials without reducing the materials’ performance
• no undesirable release during incorporation (this does not apply to resorbable products)

NanoMed

NanoMED – Toxicological characterisation of nanomaterials for the diagnostic imaging in medicine

The main task of the project NanoMed was to determine the internal exposure of novel (magnetic) nanoparticles which were specifically designed to have excellent imaging properties for a later usage in medical imaging procedures like computer tomography (CT) and magnetic resonance imaging (MRI). Particular attention was also paid to the nanoparticles’ behaviour at the blood-placenta barrier (BPB) and the blood-brain barrier (BBB) for in vitro and in vivo exposure scenarios. Since regulatory standards for the toxicity testing of nanoparticles were missing in general, all applied model test systems were systematically validated for a broad and standardised application.

The work plan included the following endpoints:

• systematic determination of nanoparticle characteristics
• development of a set of reference particles
• interactions of nanoparticles with the blood-placenta barrier (BPB) and the blood-brain barrier (BBB) in in vitro und in vivo experiments
• establishment of structure-activity relationships & dose-response relationships
• risk analysis of nanoparticles
• correlation of in vitro– and in vivo-tests
• validation of model test systems
• development of standard operation procedures (SOP)
• development of a data base to collect and manage the results achieved

The project generated a collection of nanoparticles that were based on iron, gold and silver cores and then coated with various shell materials. Next those generated nanoparticles were identified that were suitable for biological applications due to their physical, chemical and imaging properties. With the help of these specifically designed and selected nanoparticles the NanoMed project was able to establish a quality-controlled test system to evaluate the toxicological properties of the nanoparticles, standard operation procedures (SOPs) were being prepared and reference particles selected. Based on these findings biologically relevant barriers were confronted in vitro and in vivo with the selected nanoparticles and the nanoparticle-tissue effects were measured with physical, chemical and immunohistochemical methods. The project NanoMed clearly demonstrated that the observed toxic effects from in vitro tests correlated well with those obtained during in vivo experiments making these predictable for some specifications. These results will lead to a substantial progress in developing nanoparticles specifically optimised for application in medical imaging. The comprehensive data collection of nanoparticles and their properties are organised and stored within a project data base and the main findings will be transferred in the Knowledge Base Nanomaterials – DaNa.

NanoExpo

NanoExpo – Nanobalance detectors for individual-related measure-ments of nanoparticle exposures

The practical assessment of exposure to nanoscale aerosols that are released from synthetic materials requires a novel measurement technology based on easy-to-operate, robust sensors that are ready for use instantly after switch-on. Since currently suitable nanoparticle detectors for continuous personal and mobile measurements of workplace exposure to synthetic nanoparticles (NP) are not available at an acceptable price of less than 500€, the realisation of this approach calls for a compromise between sensor performance and sensor price.

Within the NanoExpo joint project a cantilever nanoparticle detector “Cantor” was developed that combines a microcontroller-controlled MEMS cantilever resonance balance with an electrophoretic nanoparticle separator. Determination of nanoparticle mass concentrations by means of Cantor is much faster than by means of conventional filter sampling and weighing. An upstream membrane filter or impactor ensures that no microparticles get into the measuring cell.

After calibration using the FMPS (fast mobility particle sizer), a measurement uncertainty of less than ±15% within a measurement range of 0-50µg/m³, and a detection limit of 6.5µg/m³ are achieved for carbon nanoparticles. The sampling time per measurement point is 2.4 min. The error of measurement remains at <15% in the case of variations in environmental conditions such as temperature (ΔT<1°C), humidity (ΔrF <10 %), and pressure (Δp<1kPa). The completely integrated setup with air intake, control and read-out electronics as well as LCD display weighs less than 400g. The life of the battery during continuous operation exceeds an eight-hour working shift.

The expected price of the device is particularly attractive: Due to the fact that the MEMS-manufactured cantilever balance is the only more expensive component, whereas the remaining components are standard parts, the component costs amount to only 200€. Considerable price reductions can be achieved by manufacturing larger quantities.

In an air-conditioned measurement chamber, comprehensive Cantor tests were carried out on carbon, TiO₂ and SiO₂ aerosols and cigarette smoke and e-cigarette vapour were analysed in the laboratory. After testing, Cantor can be easily dismantled and regenerated. Upon further optimisation of the cantilever balance and evaluation circuitry, rapid improvement is expected for parameters with a detection limit below 1µg/m³ and a sampling time of less than 1min as well as for weight and volume of the measurement cell.

DaNa

DaNa – Acquisition, evaluation and public-oriented presentation of society-relevant data and findings relating to nanomaterials

DaNa is an umbrella project aiming at collecting and evaluating scientific results in an interdisciplinary approach with scientists from different research areas, such as human and environmental toxicology, biology, physics, chemistry, and sociology. Research findings from the field of human and environmental nanotoxicology will be prepared and presented together with material properties and possible applications for interested laymen and stakeholders. This data will be published on a website and also via other media. Furthermore the scientific results of the project cluster NanoCare (NanoCare, INOS and TRACER), along with data from literature and of current and recent projects provide a basis for a critical but balanced evaluation of nanomaterials and their toxicological properties. All data will be evaluated with regard to their scientific value prior to publishing.

Pupils‘ focus groups on “Nanotechnology and environmental protection – chances and risks” will take place in three German schools within the framework of the DaNa project. They will provide pupils with the opportunity to form their own knowledge-based opinion on chances and risks associated with nanotechnological processes in the environmental sector.

CarboTox

CarboTox – Development of screening methods to analyse cancerogenous potential of carbon nano tubes

As part of the joint project CarboTox, tailor-made multi-walled carbon nanotubes (MWCNT) with different lengths, diameters and shapes, produced by our cooperation partner (IFW, Dresden), were tested both in vivo and in vitro for their toxic and carcinogenic potential.

A carcinogenicity study was performed to evaluate the carcinogenic potential of multi-walled CNT over a test duration of 2 years. To this end, rats were treated once with the test item (2 dose groups with 50 animals/group: 1 x 109 and 5 x 109 WHO fibers*/rat, amosite as reference fiber, dose: 0.1 x 109 WHO fibers*/rat) by intraperitoneal injection. In parallel, a screening test was developed with the aim to identify markers for chronic adverse effects detectable already after shorter observation periods. All multi-walled carbon nanotubes, independent of their length and diameter, caused tumors (mesothelioma) typically associated with fibers after intraperitoneal injection, but multi-walled carbon nanotubes with more needle-like morphology showed a stronger tumorigenic effect than the coiled carbon nanotubes.

*WHO fibers: length > 5 µm, diameter < 3 µm; length/diameter ratio > 3/1

Measuring the thickness of the diaphragm covering the peritoneum was found to be a well suited endpoint for an in vivo screening test with observation periods of 3 and 6 months. Equally valid is the determination of the ratio of proliferated cells compared to non-proliferated cells in the peritoneum using the BrdU method (chem. Bromodeoxyuridine). An increase in proliferated cells indicates the beginning of possibly uncontrolled cell division. A simultaneously performed subchronic inhalation test with the same multi-walled carbon nanotubes is aimed at detecting and quantifying the migration of multi-walled CNT to the pleura. With the currently available results, however, it is not yet possible to answer these questions

In vitro assays with primary mesothelial cell cultures showed that multi-walled CNT, depending on their morphology, inhibit cell proliferation, lead to damage of membranes, cytoskeleton and DNA, and cause cellular senescence. The strongest biological effects were induced by multi-walled carbon nanotubes with needle-like morphology.

Interestingly, the effects of different multi-walled carbon nanotubes observed in cell cultures correlated with tumor development in rats, so that certain cell culture methods might be suitable for use as screening tests to assess the adverse, carcinogenic potential of new multi-walled CNT.