Final Report Summary - NANONEUROHOP (Assessment of the hazard and opportunities of using carbon nanotubes as a new nanocarrier for drug delivery in neural tissue)
Objectives
The overall scientific objective of the project was to understand better the interaction between carbon nanotubes, nanoscaled tubes made of carbon atoms, and the brain in order to improve their biocompatibility for this tissue.
The first specific objective was to investigate the toxicological profile of different functionalized carbon nanotubes, in the context of their use as nanovectors for drug/gene delivery in the brain. The host lab has indeed demonstrated in proof-of-concept studies that carbon nanotubes directly injected in the brain were promising vectors of nucleic acid sequences to alleviate brain disorders, such as stroke. In this context, the aim was to understand better the interactions of carbon nanotubes with cells populating the brain parenchyma, and to identify their potential deleterious effects.
The second objective was to assess the interactions from a different perspective, not driven by toxicology. The aim was here to evaluate over time the potential degradability of functionalized carbon nanotubes within brain tissue but also in isolated microglial cells, the resident macrophages of the brain. Taking into consideration previous data generated by the host lab, it was also intended to investigate the mobility/diffusion within the brain of individualized nanoscaled entities such as carbon nanotubes.
Finally, a second overall aim of the project was to train the funded Researcher so as to develop the managerial, writing, mentoring skills and wider scientific expertise that are needed to reach an independent position.
Work performed & main results achieved
At the beginning of the project, different models of primary cell cultures were established. They covered the main cell types that populate the brain parenchyma, namely neurons, glial cells and microglia. Following optimization, the cell cultures were prepared from two selected brain regions: the frontal cortex and the striatum. When ready, the cells were then exposed for 24 hours to increasing dose of carbon nanotubes so as to determine the dose-response relationship. Pristine and functionalized carbon nanotubes were used for that purpose. Functionalized carbon nanotubes varied according to the surface charges (negative, positive or both negative and positive).
From this study, no toxicity was observed when exposing neuron enriched cell cultures from both frontal cortex and striatum. Similarly, we did not observed significant toxicity of carbon nanotubes for mixed glial cell cultures from frontal cortex but observed toxicity for cell cultures prepared from striatum; and the toxicity was not significantly dose-dependent. Optical microscope observations revealed that some cells within glial cell cultures were engulfing more nanotubes than others and that those carbon nanotube loaded cells were more numerous in striatum derived cell cultures than in frontal cortex derived ones. Using immuno-staining, the carbon nanotube loaded cells were identified as microglial cells whereas most of the other cells composing the mixed glial cell cultures were astrocytes. These staining also confirmed that microglial cells were more numerous in striatum derived cell cultures than in frontal cortex derived ones. Moreover, carbon nanotubes were dose-dependently cytotoxic for isolated microglial cell cultures. Taken together, those results demonstrated that the cytotoxicity observed with mixed glial cell cultures from the striatum could be due to their cell composition, with a higher amount of microglial cells which are more sensitive to carbon nanotubes than other brain cell types.
Following on the data from the first study, the sensitivity of microglia to carbon nanotubes was investigated further. One non-cytotoxic-at-24-hours dose was selected for three types of nanotubes: positively charged, negatively charged and both positively and negatively charged. Using the long term survival and non-dividing properties of primary microglial cell cultures, the effects of carbon nanotubes on isolated microglial cells were here assessed over a period of one month after a single exposure. The long term cytotoxicity, the migration and uptake capabilities, and production of nitric oxide were assessed. Even though microglia appeared full of carbon nanotubes under optical microscope, no significant cytotoxicity, no major effects on their migration and engulfment abilities, and also no significant release of nitric oxide were demonstrated, compared to control cells non-exposed to nanotubes, at the concentration used.
Microglia which are resident macrophages of the brain are not only able to engulf foreign materials but can also process them, leading to their degradation or elimination. The potential degradability of the same three functionalized carbon nanotubes was therefore investigated with primary microglial cell cultures over a period of 3 months. Modification of the structure of carbon nanotubes that have been engulfed were found for all three different nanotubes. Importantly, carbon nanotubes were still present after 3 months, confirming that degradation is slow in microglia. For materials baring negative charges, structural modifications were highly significant just after the exposure but were slowing down thereafter. Materials baring positive charges were also undergoing modifications but with a rate similar to the second phase observed with negatively charged nanotubes (slight difference between successive time points).
Still looking after potential degradability of carbon nanotubes but this time in a more dynamic model, one type of positively charged carbon nanotubes was selected and injected into superficial brain areas of rodent brains. Two days only after injection, structural modifications of materials were already observed, continuing after 2 weeks. Going further, animals were injected in deep brain regions with the same type of nanotubes and their degradability was here assessed after up to 100 days. Diffusion and clearance mechanisms were also considered for this long term assessment. Preliminary results showed that degradation was more advanced than after 2 weeks in superficial areas, and that carbon nanotubes were found outside the brain. Importantly, for both experiments, the overall behaviour and apparent health of the animals which were injected were not affected.
Impact and use
This project is directly addressing the societal question of developing nanomaterials for specific applications while also considering their safety so as to provide the best benefit for human health and minimize the risk.
The results obtained during these two years have improved the understanding of the host lab on the reaction of the brain when expose to carbon nanotubes, highlighting the key role of microglia. It will help the host lab toward the design of more biocompatible materials (taking into consideration not only the materials but also the brain regions where to implant those drug nanovectors). These kinds of specific toxicological studies dedicated to the tissue where the biomaterials might be used for biomedical applications are important toward the clinical development of carbon nanotubes, or any other nanomaterials, as medical solutions.
Further investigations will of course be needed to answer some questions that have been raised during the course of the project. Due to the lack of knowledge and some inconsistency in previous findings, studies were mainly focused on short term effects (below 3 months). But long term studies will be required in order to explain the apparent biocompatibility of carbon nanotubes in brain and to assess the long term fate (safety and degradation) of nanotubes after parenchymal injection.
The training of the experienced Researcher toward an independent position was fruitful. About eight less experienced scientists have benefited directly from his mentoring at some point during their projects. Overall, about twelve peer-reviewed publications and two book chapters will be generated from these two years. Finally, the Researcher was offered a lectureship during the last few months of the project and will start soon his independent carrier, and may continue some studies started during the project.
The overall scientific objective of the project was to understand better the interaction between carbon nanotubes, nanoscaled tubes made of carbon atoms, and the brain in order to improve their biocompatibility for this tissue.
The first specific objective was to investigate the toxicological profile of different functionalized carbon nanotubes, in the context of their use as nanovectors for drug/gene delivery in the brain. The host lab has indeed demonstrated in proof-of-concept studies that carbon nanotubes directly injected in the brain were promising vectors of nucleic acid sequences to alleviate brain disorders, such as stroke. In this context, the aim was to understand better the interactions of carbon nanotubes with cells populating the brain parenchyma, and to identify their potential deleterious effects.
The second objective was to assess the interactions from a different perspective, not driven by toxicology. The aim was here to evaluate over time the potential degradability of functionalized carbon nanotubes within brain tissue but also in isolated microglial cells, the resident macrophages of the brain. Taking into consideration previous data generated by the host lab, it was also intended to investigate the mobility/diffusion within the brain of individualized nanoscaled entities such as carbon nanotubes.
Finally, a second overall aim of the project was to train the funded Researcher so as to develop the managerial, writing, mentoring skills and wider scientific expertise that are needed to reach an independent position.
Work performed & main results achieved
At the beginning of the project, different models of primary cell cultures were established. They covered the main cell types that populate the brain parenchyma, namely neurons, glial cells and microglia. Following optimization, the cell cultures were prepared from two selected brain regions: the frontal cortex and the striatum. When ready, the cells were then exposed for 24 hours to increasing dose of carbon nanotubes so as to determine the dose-response relationship. Pristine and functionalized carbon nanotubes were used for that purpose. Functionalized carbon nanotubes varied according to the surface charges (negative, positive or both negative and positive).
From this study, no toxicity was observed when exposing neuron enriched cell cultures from both frontal cortex and striatum. Similarly, we did not observed significant toxicity of carbon nanotubes for mixed glial cell cultures from frontal cortex but observed toxicity for cell cultures prepared from striatum; and the toxicity was not significantly dose-dependent. Optical microscope observations revealed that some cells within glial cell cultures were engulfing more nanotubes than others and that those carbon nanotube loaded cells were more numerous in striatum derived cell cultures than in frontal cortex derived ones. Using immuno-staining, the carbon nanotube loaded cells were identified as microglial cells whereas most of the other cells composing the mixed glial cell cultures were astrocytes. These staining also confirmed that microglial cells were more numerous in striatum derived cell cultures than in frontal cortex derived ones. Moreover, carbon nanotubes were dose-dependently cytotoxic for isolated microglial cell cultures. Taken together, those results demonstrated that the cytotoxicity observed with mixed glial cell cultures from the striatum could be due to their cell composition, with a higher amount of microglial cells which are more sensitive to carbon nanotubes than other brain cell types.
Following on the data from the first study, the sensitivity of microglia to carbon nanotubes was investigated further. One non-cytotoxic-at-24-hours dose was selected for three types of nanotubes: positively charged, negatively charged and both positively and negatively charged. Using the long term survival and non-dividing properties of primary microglial cell cultures, the effects of carbon nanotubes on isolated microglial cells were here assessed over a period of one month after a single exposure. The long term cytotoxicity, the migration and uptake capabilities, and production of nitric oxide were assessed. Even though microglia appeared full of carbon nanotubes under optical microscope, no significant cytotoxicity, no major effects on their migration and engulfment abilities, and also no significant release of nitric oxide were demonstrated, compared to control cells non-exposed to nanotubes, at the concentration used.
Microglia which are resident macrophages of the brain are not only able to engulf foreign materials but can also process them, leading to their degradation or elimination. The potential degradability of the same three functionalized carbon nanotubes was therefore investigated with primary microglial cell cultures over a period of 3 months. Modification of the structure of carbon nanotubes that have been engulfed were found for all three different nanotubes. Importantly, carbon nanotubes were still present after 3 months, confirming that degradation is slow in microglia. For materials baring negative charges, structural modifications were highly significant just after the exposure but were slowing down thereafter. Materials baring positive charges were also undergoing modifications but with a rate similar to the second phase observed with negatively charged nanotubes (slight difference between successive time points).
Still looking after potential degradability of carbon nanotubes but this time in a more dynamic model, one type of positively charged carbon nanotubes was selected and injected into superficial brain areas of rodent brains. Two days only after injection, structural modifications of materials were already observed, continuing after 2 weeks. Going further, animals were injected in deep brain regions with the same type of nanotubes and their degradability was here assessed after up to 100 days. Diffusion and clearance mechanisms were also considered for this long term assessment. Preliminary results showed that degradation was more advanced than after 2 weeks in superficial areas, and that carbon nanotubes were found outside the brain. Importantly, for both experiments, the overall behaviour and apparent health of the animals which were injected were not affected.
Impact and use
This project is directly addressing the societal question of developing nanomaterials for specific applications while also considering their safety so as to provide the best benefit for human health and minimize the risk.
The results obtained during these two years have improved the understanding of the host lab on the reaction of the brain when expose to carbon nanotubes, highlighting the key role of microglia. It will help the host lab toward the design of more biocompatible materials (taking into consideration not only the materials but also the brain regions where to implant those drug nanovectors). These kinds of specific toxicological studies dedicated to the tissue where the biomaterials might be used for biomedical applications are important toward the clinical development of carbon nanotubes, or any other nanomaterials, as medical solutions.
Further investigations will of course be needed to answer some questions that have been raised during the course of the project. Due to the lack of knowledge and some inconsistency in previous findings, studies were mainly focused on short term effects (below 3 months). But long term studies will be required in order to explain the apparent biocompatibility of carbon nanotubes in brain and to assess the long term fate (safety and degradation) of nanotubes after parenchymal injection.
The training of the experienced Researcher toward an independent position was fruitful. About eight less experienced scientists have benefited directly from his mentoring at some point during their projects. Overall, about twelve peer-reviewed publications and two book chapters will be generated from these two years. Finally, the Researcher was offered a lectureship during the last few months of the project and will start soon his independent carrier, and may continue some studies started during the project.