Final Report Summary - BRASINOEU (Translocation and Safe Design of Surface Engineered Metal oxide Nanoparticles)
Translocation and Safe Design of Surface Engineered Metal Oxide Nanoparticles (BRASINOEU)
Launched in January 2013. Duration 4 years
BRASINOEU is an EU-funded research project in the framework of FP7 - Marie Curie Actions PIRSES (Grant Agreement Number: PIRSES-GA-2012-318916).
Coordinator of the Project: Dr. I. Estrela-Lopis, University of Leipzig, Germany,
Irina.Estrela-Lopis@medizin.uni-leipzig.de
http://www.uni-leipzig.de/~biophys/Irina/Estrela_BRASINOEU_Homepage.html
Partners: CIC biomaGUNE, Spain; Finnish Institute of Occupational Health, Finland; Institute of Physics of Polish Academy of Sciences, Poland (IPPAS);Medical University of Warsaw, Poland; Zhejiang University, China; University of Brasilia, Brasilia; Huazhong University of Science and Technology, China
Scientific and technological developments of nanotechnology require a deeper knowledge of the effects of nanotechnology based products on human health. This knowledge is fundamental for the development of nanotechnology and to achieve its full acceptance by the society. In this context the concept of "safe by design" means to design the nanomaterials in such a manner that their possible harm to humans and the environment are prevented or at least reduced. Within BRASINOEU the translocation and nanosafety issues of engineered metal oxide nanoparticles (NPs) have been studied. The project has encompassed the synthesis of metal oxide NPs, their surface modification and post modification in biological fluids; immunological and genotoxicity studies, and translocation studies both "in vitro" and "in vivo". The correlations between designed NP properties and their translocation at cellular and body level as well as their immune- and genotoxic response have been studied. These correlations are also pivotal for the classification of NPs according to their toxicological impact and for the application of the safe-by-design approach to the development of NPs and devices utilizing them to avoid risks to human health and the environment. State of the art high resolution techniques have been employed for studying localization and quantification of NPs, such as transmission electron microscopy (TEM), confocal Raman microscopy (CRM), and ion beam microscopy (IBM). Positron emission tomography (PET) and single-photon emission computed tomography (SPECT) have been employed for biodistribution and quantification studies in animal models. Description of main results.
The NPs with well-defined properties and chemistry were synthesized and characterized. A key aspect of the project was to tune the surface chemistry of the NPs to find ways to increase biocompatibility and to control NP translocation at cellular and body level. Pristine, citrate, dimercaptosuccinic acid, alkyl, dye, brush etc. modified maghemite, Au and CeO2-x NPs were synthesized and thoroughly characterized by means of state of the art techniques in dry state as well in biological fluids. The NPs with the “best” properties, regarding nano-size distribution, stability in suspension and superparamagnetic properties etc. were selected for subsequent translocation/quantification studies and toxicity assays. Additionally NPs with a high potential for biomedical and industrial applications were designed:
a) Superparamagnetic iron oxide nanoparticles (SPIONs) functionalized with pH sensitive biopolymers as potential nanostructures for transcutaneous drug delivery.
b) SPIONs with designed magnetic properties as a tool for temperature measurements with a high precision and for temperature control in tumor hyperthermia treatment
c) Nanocosructs doped with Yb, Tm and Er combining an intrinsic fluorescence, photosensitization of reactive oxygen species (ROS) and magnetic/superparamagnetic properties. These novel nanodevices could thus be applied in combined magnetic hyperthermia and photodynamic therapy of cancer patients.
d) Selol loaded magnetic PLGA particles containing iron oxide as an effective system for magnetically controlled drug delivery and as a magnetohyperthermia inductor in antitumor therapy. On the basis of the results concerning the selol loaded particles several medical institutions in Poland have acquired a permission to carry out pilot clinical studies on cancer cases of patients excluded from further therapy.
Spatially resolved dosimetric and high resolution imaging techniques as IBM, CRM, TEM, PET/SPECT were developed and tailored to quantify and to visualize the translocation pattern of NPs in biological media, culture cells and in ex-vivo. The main conclusions of translocation studies are:
1. The chain length of the ligands plays an important role in many biomedical applications. The interaction of nanoparticles carrying surface-capped ligands of different length, in particular the hydrophobic chain length, with cells is of major importance for understanding the mechanism of cellular uptake and toxicity. Translocation and uptake quantification studies showed that the length of the hydrophobic part of the surface coating of NPs as well as the adsorption of adhesion mediators on NP surface are crucial for the interactions with proteins and cells and as consequence the uptake rate. These findings allowed tuning the uptake and reducing the adverse effects, as well as achieving a significant therapeutic impact.
2. Apart from the stabilizing function of polymer brushes on the surface of NPs, the brushes had the following additional functions: i) to tune the uptake of NPs as a function of surface charge, polymer chain length and surface chemical composition; ii) hide and /or present the “authentic” surface of NPs bearing different oxidation states and oxygen defects as ‘hot spots’ for catalytic reactions; iii) hide fluorescence groups within brushes without altering the surface properties for fast diagnostic applications. The labelling of NPs affected strongly their aggregation state, uptake kinetics and intracellular co-localization pattern at single cell level. Only short polymer brushes on the surface of NPs were capable of shielding the dye reporter molecules within the polymer layer. Label-free uptake studies in vitro revealed that polymer brushes with different charges decreased the uptake of CeO2-x NPs. Furthermore, the capacity to scavenge ROS and to protect cells from oxidative stress based on redox properties of CeO2-x surface was affected by polymer brushes. 60 % and 7% of the original scavenging capacity remained for negatively and positively charged polymer brushes, respectively. These results correlated well with the in vivo (Female C57BI/6 mice) immune and genotoxic responses of pristine CeO2-x NPs and those modified with negatively and positively charged polymer brushes. The results showed that functionalization of NPs with positively charged polymer brushes clearly induced an immunological response in mice after repeated administration of the NPs by intraperitoneal route.
3. Uptake studies conducted with pristine maghemite NPs showed a very high average intracellular Fe concentration compared to citrate modified NPs. This Fe value was up to several orders of magnitude higher than the intrinsic iron and phosphor concentration in control culture cells. Furthermore, the applied pristine maghemite NPs had a significant impact on metabolically relevant intrinsic and trace elements. This finding correlated with the genotoxic assays of maghemite NPs. Pristine maghemite NPs are able to induce both DNA and chromosomal damage in cell cultures and have the potential to induce permanent genetic damage. Conversely, exposure to citrate-coated maghemite NPs, resulted in a lower intracellular concentration of NPs and an absence of ability to induce DNA damage at any of the exposure times, suggesting that the coating could prevent their genotoxic effect.
4. The double labeling strategy with two gamma emitters, one located in the core (111In) and the other in the shell (125I) were developed for tracing the biological fate of NPs at the organism level in vivo. The degradation kinetic of core-shell constructs and release of iron oxide NPs after they were delivered to the target organs by carrier particles were visualized and quantified.
The obtained knowledge on the impact of surface modification, such as citrate, brush and alkyl on uptake rate and in general on toxicity can be used as an effective tool to control the uptake for medical applications, risk assessment and risk mitigation. Additionally, the strategy of hiding reporter molecules within capped stabilized layers without altering the surface properties could be implemented for fast and cost-effective screening, monitoring and diagnostic purposes. The knowledge on biodistribution and fate in in vivo is very important for the future use of NPs as contrast agents, which is of high economic importance. Some of the radio labelled NPs can find their way into the market as contrast agents for PET or SPECT, and have therefore a medical and economic significance in themselves. The knowledge of bio-kinetics by means of dual labelling is pivotal to understand how to engineer the NP for reaching specific organs and how to control their fate in body circulation, which is a crucial issue in delivery. This will have a direct impact on the pharmacological industry and in the design of solutions for drug delivery based on NPs. This knowledge will promote the generation of NPs capable of targeting selected organs that can be applied in small doses with a high efficiency. This will have an enormous economic impact since such solutions are required for diseases such as arthritis, cancer, atherosclerosis to cite a few.
The knowledge concerning the biological responses in in vivo and in vitro could be used to diminish the risks of the NPs by selecting those surface modifications that pose minimal danger. However, vitro-vivo relevance as well as the predictive potential of a short-term for a long-term in vivo experiments are still a critical issues in nanotoxicology that should be addressed for developing risk assessment tools, reduction an eventual risk of NPs on human and environment as well as to promote industrial development of the nanotechnology sector.
Launched in January 2013. Duration 4 years
BRASINOEU is an EU-funded research project in the framework of FP7 - Marie Curie Actions PIRSES (Grant Agreement Number: PIRSES-GA-2012-318916).
Coordinator of the Project: Dr. I. Estrela-Lopis, University of Leipzig, Germany,
Irina.Estrela-Lopis@medizin.uni-leipzig.de
http://www.uni-leipzig.de/~biophys/Irina/Estrela_BRASINOEU_Homepage.html
Partners: CIC biomaGUNE, Spain; Finnish Institute of Occupational Health, Finland; Institute of Physics of Polish Academy of Sciences, Poland (IPPAS);Medical University of Warsaw, Poland; Zhejiang University, China; University of Brasilia, Brasilia; Huazhong University of Science and Technology, China
Scientific and technological developments of nanotechnology require a deeper knowledge of the effects of nanotechnology based products on human health. This knowledge is fundamental for the development of nanotechnology and to achieve its full acceptance by the society. In this context the concept of "safe by design" means to design the nanomaterials in such a manner that their possible harm to humans and the environment are prevented or at least reduced. Within BRASINOEU the translocation and nanosafety issues of engineered metal oxide nanoparticles (NPs) have been studied. The project has encompassed the synthesis of metal oxide NPs, their surface modification and post modification in biological fluids; immunological and genotoxicity studies, and translocation studies both "in vitro" and "in vivo". The correlations between designed NP properties and their translocation at cellular and body level as well as their immune- and genotoxic response have been studied. These correlations are also pivotal for the classification of NPs according to their toxicological impact and for the application of the safe-by-design approach to the development of NPs and devices utilizing them to avoid risks to human health and the environment. State of the art high resolution techniques have been employed for studying localization and quantification of NPs, such as transmission electron microscopy (TEM), confocal Raman microscopy (CRM), and ion beam microscopy (IBM). Positron emission tomography (PET) and single-photon emission computed tomography (SPECT) have been employed for biodistribution and quantification studies in animal models. Description of main results.
The NPs with well-defined properties and chemistry were synthesized and characterized. A key aspect of the project was to tune the surface chemistry of the NPs to find ways to increase biocompatibility and to control NP translocation at cellular and body level. Pristine, citrate, dimercaptosuccinic acid, alkyl, dye, brush etc. modified maghemite, Au and CeO2-x NPs were synthesized and thoroughly characterized by means of state of the art techniques in dry state as well in biological fluids. The NPs with the “best” properties, regarding nano-size distribution, stability in suspension and superparamagnetic properties etc. were selected for subsequent translocation/quantification studies and toxicity assays. Additionally NPs with a high potential for biomedical and industrial applications were designed:
a) Superparamagnetic iron oxide nanoparticles (SPIONs) functionalized with pH sensitive biopolymers as potential nanostructures for transcutaneous drug delivery.
b) SPIONs with designed magnetic properties as a tool for temperature measurements with a high precision and for temperature control in tumor hyperthermia treatment
c) Nanocosructs doped with Yb, Tm and Er combining an intrinsic fluorescence, photosensitization of reactive oxygen species (ROS) and magnetic/superparamagnetic properties. These novel nanodevices could thus be applied in combined magnetic hyperthermia and photodynamic therapy of cancer patients.
d) Selol loaded magnetic PLGA particles containing iron oxide as an effective system for magnetically controlled drug delivery and as a magnetohyperthermia inductor in antitumor therapy. On the basis of the results concerning the selol loaded particles several medical institutions in Poland have acquired a permission to carry out pilot clinical studies on cancer cases of patients excluded from further therapy.
Spatially resolved dosimetric and high resolution imaging techniques as IBM, CRM, TEM, PET/SPECT were developed and tailored to quantify and to visualize the translocation pattern of NPs in biological media, culture cells and in ex-vivo. The main conclusions of translocation studies are:
1. The chain length of the ligands plays an important role in many biomedical applications. The interaction of nanoparticles carrying surface-capped ligands of different length, in particular the hydrophobic chain length, with cells is of major importance for understanding the mechanism of cellular uptake and toxicity. Translocation and uptake quantification studies showed that the length of the hydrophobic part of the surface coating of NPs as well as the adsorption of adhesion mediators on NP surface are crucial for the interactions with proteins and cells and as consequence the uptake rate. These findings allowed tuning the uptake and reducing the adverse effects, as well as achieving a significant therapeutic impact.
2. Apart from the stabilizing function of polymer brushes on the surface of NPs, the brushes had the following additional functions: i) to tune the uptake of NPs as a function of surface charge, polymer chain length and surface chemical composition; ii) hide and /or present the “authentic” surface of NPs bearing different oxidation states and oxygen defects as ‘hot spots’ for catalytic reactions; iii) hide fluorescence groups within brushes without altering the surface properties for fast diagnostic applications. The labelling of NPs affected strongly their aggregation state, uptake kinetics and intracellular co-localization pattern at single cell level. Only short polymer brushes on the surface of NPs were capable of shielding the dye reporter molecules within the polymer layer. Label-free uptake studies in vitro revealed that polymer brushes with different charges decreased the uptake of CeO2-x NPs. Furthermore, the capacity to scavenge ROS and to protect cells from oxidative stress based on redox properties of CeO2-x surface was affected by polymer brushes. 60 % and 7% of the original scavenging capacity remained for negatively and positively charged polymer brushes, respectively. These results correlated well with the in vivo (Female C57BI/6 mice) immune and genotoxic responses of pristine CeO2-x NPs and those modified with negatively and positively charged polymer brushes. The results showed that functionalization of NPs with positively charged polymer brushes clearly induced an immunological response in mice after repeated administration of the NPs by intraperitoneal route.
3. Uptake studies conducted with pristine maghemite NPs showed a very high average intracellular Fe concentration compared to citrate modified NPs. This Fe value was up to several orders of magnitude higher than the intrinsic iron and phosphor concentration in control culture cells. Furthermore, the applied pristine maghemite NPs had a significant impact on metabolically relevant intrinsic and trace elements. This finding correlated with the genotoxic assays of maghemite NPs. Pristine maghemite NPs are able to induce both DNA and chromosomal damage in cell cultures and have the potential to induce permanent genetic damage. Conversely, exposure to citrate-coated maghemite NPs, resulted in a lower intracellular concentration of NPs and an absence of ability to induce DNA damage at any of the exposure times, suggesting that the coating could prevent their genotoxic effect.
4. The double labeling strategy with two gamma emitters, one located in the core (111In) and the other in the shell (125I) were developed for tracing the biological fate of NPs at the organism level in vivo. The degradation kinetic of core-shell constructs and release of iron oxide NPs after they were delivered to the target organs by carrier particles were visualized and quantified.
The obtained knowledge on the impact of surface modification, such as citrate, brush and alkyl on uptake rate and in general on toxicity can be used as an effective tool to control the uptake for medical applications, risk assessment and risk mitigation. Additionally, the strategy of hiding reporter molecules within capped stabilized layers without altering the surface properties could be implemented for fast and cost-effective screening, monitoring and diagnostic purposes. The knowledge on biodistribution and fate in in vivo is very important for the future use of NPs as contrast agents, which is of high economic importance. Some of the radio labelled NPs can find their way into the market as contrast agents for PET or SPECT, and have therefore a medical and economic significance in themselves. The knowledge of bio-kinetics by means of dual labelling is pivotal to understand how to engineer the NP for reaching specific organs and how to control their fate in body circulation, which is a crucial issue in delivery. This will have a direct impact on the pharmacological industry and in the design of solutions for drug delivery based on NPs. This knowledge will promote the generation of NPs capable of targeting selected organs that can be applied in small doses with a high efficiency. This will have an enormous economic impact since such solutions are required for diseases such as arthritis, cancer, atherosclerosis to cite a few.
The knowledge concerning the biological responses in in vivo and in vitro could be used to diminish the risks of the NPs by selecting those surface modifications that pose minimal danger. However, vitro-vivo relevance as well as the predictive potential of a short-term for a long-term in vivo experiments are still a critical issues in nanotoxicology that should be addressed for developing risk assessment tools, reduction an eventual risk of NPs on human and environment as well as to promote industrial development of the nanotechnology sector.