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Projects / Programmes source: ARIS

Evaluation of possible harmful effects of nanoparticles and underlying mechanisms - from physico-chemical and in vitro toxicity characterisation to innate immune system activation

Research activity

Code Science Field Subfield
7.00.00  Interdisciplinary research     

Code Science Field
B000  Biomedical sciences   

Code Science Field
2.10  Engineering and Technology  Nano-technology 
Keywords
nanoparticles, immunotoxicity, native immune system, protein corona, physico-chemical characterisation
Evaluation (rules)
source: COBISS
Researchers (23)
no. Code Name and surname Research area Role Period No. of publicationsNo. of publications
1.  36454  PhD Maruša Bizjak  Biochemistry and molecular biology  Junior researcher  2016 - 2018  31 
2.  37546  PhD Janja Božič  Medical sciences  Researcher  2018  30 
3.  19411  PhD Vladimir Boštjan Bregar  Materials science and technology  Head  2016 - 2018  105 
4.  28476  PhD Nataša Drnovšek  Materials science and technology  Researcher  2016 - 2018  87 
5.  52538  Jan Gregorec  Biotechnology  Researcher  2018 
6.  06628  PhD Roman Jerala  Biochemistry and molecular biology  Researcher  2016 - 2018  1,189 
7.  18619  PhD Maša Kandušer  Pharmacy  Researcher  2018  160 
8.  00412  PhD Igor Križaj  Biochemistry and molecular biology  Researcher  2016 - 2018  725 
9.  18802  PhD Adrijana Leonardi  Biochemistry and molecular biology  Researcher  2016 - 2018  156 
10.  34385  PhD Jasna Lojk  Metabolic and hormonal disorders  Researcher  2016 - 2017  68 
11.  34138  PhD Martina Lorenzetti  Materials science and technology  Researcher  2016  54 
12.  39583  Petra Malavašič  Biochemistry and molecular biology  Technical associate  2016 - 2017  32 
13.  21426  PhD Mateja Manček Keber  Pharmacy  Researcher  2016 - 2018  159 
14.  04292  PhD Saša Novak Krmpotič  Materials science and technology  Researcher  2016 - 2018  668 
15.  26163  PhD Gabriela Panter  Biotechnology  Researcher  2016  45 
16.  19225  PhD Mojca Pavlin  Systems and cybernetics  Researcher  2016 - 2018  262 
17.  37589  PhD Tina Tinkara Peternelj  Microbiology and immunology  Researcher  2016  39 
18.  30710  PhD Sonja Prpar Mihevc  Neurobiology  Beginner researcher  2016 - 2017  108 
19.  15813  PhD Boris Rogelj  Neurobiology  Researcher  2016 - 2018  412 
20.  37508  PhD Klemen Strojan  Biotechnology  Researcher  2016 - 2018  25 
21.  38850  PhD Nives Škorja Milić  Neurobiology  Technical associate  2016  17 
22.  10974  PhD Irena Zajc  Biochemistry and molecular biology  Technical associate  2017  134 
23.  51691  Ažbe Žnidaršič    Technical associate  2018 
Organisations (3)
no. Code Research organisation City Registration number No. of publicationsNo. of publications
1.  0104  National Institute of Chemistry  Ljubljana  5051592000  20,916 
2.  0106  Jožef Stefan Institute  Ljubljana  5051606000  90,600 
3.  1538  University of Ljubljana, Faculty of Electrical Engineering  Ljubljana  1626965  27,738 
Abstract
Despite the ever expanding use and exposure to nanoparticles (NPs), there are still several open questions concerning NP safety. Recently, immunotoxicity and immunogenicity of NPs have emerged as an important problem and for several types of NPs, especially engineered NPs, the risks have not been adequately assessed. Also, due to the complexity and individual variability of the immune system (IS) as well as the vast diversity of NPs, the interactions between the NPs and the IS are not well understood. Several studies showed the importance of detailed characterization of NPs and assessment of their properties in the physiologically relevant media and the protein corona for better understanding of NP toxicity. However, several recent papers also stressed the necessity for better assessment of nanoimmunosafety as there is still a limited understanding of the connection between the physicochemical properties of NPs and only a few studies analysed the NP corona in relation to IS activation by NPs. In this project, we will perform detailed physicochemical characterization of our biomedical (PAA and PEI coated) and selected engineered NPs (TiO2, SiO2, Ag). We will also analyse the presence of impurities and other contaminations of the NPs solutions, which might impact the immune system (e.g. endotoxins). Next, we will analyse the protein corona, formed on the surface of NPs in different physiological conditions (different serum concentrations, comparison of human and bovine serum), with the focus on proteins relevant for the immune system, such as the components of the complement system and antibodies. Also we will examine the ability of the NPs to activate the complement system, which is well defined only for a few biomedical NPs.  The adsorption of molecules on the NP surface critically determines if and how the NPs will interact with the cells and triggers the immune system. The interactions between NPs and cells of the innate IS  (primary human macrophages, neutrophils) will be observed on a morphological (mechanisms of uptake and intracellular fate, uptake quantification) and physiological level (cell activation and maturation, cytokine secretion, activation of key transcription factors). We will also investigate the impact of NP exposure on the ability of the cells of the innate IS to perform their function. The collaboration between leading Slovenian groups in each field will enable us to connect the results into a comprehensive analysis of the interactions between NPs and the innate IS: from thorough NP characterization, the composition of the protein corona, to the interactions between NPs and innate IS cells and their consequences. This knowledge will enable us to assess the possible harmful effect of NP on the cells of the innate IS and help us implement the ‘safer by design’ principle for design of new NPs.
Significance for science
In this project proposal we aim to combine transdisciplinary approach, from standard physicochemical characterisation and in vitro toxicity assays to more advanced chemical analysis of material, biochemical characterization of protein corona combined with analysis of possible innate immune activation. This was also identified by the EU commission, which prompts assessment of the immunogenicity and immunotoxicity of NPs. Moreover, not many studies have determined the effect of NPs on the immune system, so the mechanisms thorough which NPs interfere with immune system are not well understood.   NPs interaction with physiological media alters their stability (effective size) and effective zeta potential (charge) and consequently their toxicity and immunogenicity, – so characterisation in physiological media is crucial. The analysis of protein corona is also crucial, since NPs interaction with absorbed proteins can trigger their conformational changes that can in turn stimulate the innate immune system receptors. Connected to WP2 and WP3, activation of the complement system, crucial part of innate IS, will be analysed. NPs can also alter immune cells functions or can have a direct toxic effect on macrophages and other cells of IS – therefore a set of in vitro experiments will evaluate possible effects on cells from morphological changes, cell viability and proliferation to measurements of relevant cytokines.   There are many studies relating to physicochemical characterisation, in vitro toxicity assays, chemical analysis of material, biochemical characterization of protein corona, or analysis of potential immune activation separately, while studies combining physicochemical characterisation, protein corona determination and innate immune system activation are extremely rare. Our goal is to connect all those fields inside of one study for each of the different NPs to be evaluated; this will enable better understanding of the mechanisms responsible for immunotoxicity of NPs.   Too properly asses’ potential nontoxicity, it is imperative that materials are free of toxic contaminants that might confound the results. By carefully determining the presence of any chemical impurities and endotoxins, we will point out the relevance of such experiments before addressing immunotoxicity studies.   Executing this project will enable us to:   Contribute to the assessment of possible harmful effects and activation of the innate immune system due to exposure to different NPs. Connect physicochemical characterisation, in vitro toxicity assays, chemical analysis of material, biochemical characterization of protein corona, and analysis of possible innate immune activation to a rational whole. Analyse the mechanisms of the innate immune system activation by the NPs Implement the “safer by design” principle for designing of new NPs.
Significance for the country
This project will generate new knowledge on the interactions between nanoparticles and different cells of the innate immune system in relation to physicochemical properties of nanoparticles and the in vivo relevant protein corona. We will also explore the connection between nanoparticles surface properties and the properties of the obtained protein corona, which will help us understand the processes of nanoparticles stability and destabilization in different physiological media, like cell culture media or blood, which crucially determines NP uptake and their ability to cross the barriers, and consequently also their toxicity. Moreover, we will compare the results obtained on stable cell lines to those on primary human cells, as well as the effect of bovine and human serum, to evaluate the relevance of in vitro obtained data for in vivo transition. The knowledge obtained with the proposed project can be used to develop safer nanoparticles, nanocarriers and other nanomaterials, which do not trigger the immune system, or trigger it in a desired and controlled way. This knowledge can be applied in different biotechnological and pharmaceutical companies as well as industry.     In collaboration with complementary research groups from nanotechnology, immunology, cell biology, chemistry, biotechnology, we will establish a set of protocols for analysis and testing of nanoparticles: from physicochemical characterization, analysis of impurities, analysis of the protein corona and analysis of interactions between nanoparticles and cells. Such complete analysis has so far not been possible in Slovenia and we, as well as other interested research groups or industry, will be able to use this knowledge and protocols to test new formulations of nanoparticles. These thorough analyses, testing of nanotoxicity and immunotoxicity are essential for implementation of any new nanoparticle based application and could serve as industry guidelines. Better understanding of mechanisms together with testing will improve the evaluation of new nanomaterials industrial applications in the design stage and greatly improve the safety of industrial nanomaterials before they reach the market.     Moreover, european commission identified nanoparticle risk assessment as a potential problem and prompts assessment of the immunogenicity and immunotoxicity of NPs. This project represents a potential basis for a possible European project since we will obtain new knowledge and develop protocols necessary for a thorough examination of innate immune cells responses to nanoparticles, which can be used also as guidelines for industry for testing of new nanomaterials before they reach the market or environment. Assessment and prevention of NP toxicity and immunogenicity is also essential for public health and environment protection. Also, the new knowledge could help us establish protocols for quick testing of immunotoxicity and immunogenicity of nanoparticles.
Most important scientific results Interim report, final report
Most important socioeconomically and culturally relevant results Interim report, final report
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