University of Groningen Fluorescent Nanodiamonds as Free Radical Sensors in Aging Yeast Cells van der Laan, Kiran

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Fluorescent Nanodiamonds as Free Radical Sensors in Aging Yeast Cells

van der Laan, Kiran

DOI:

10.33612/diss.112906297

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Publication date: 2020

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van der Laan, K. (2020). Fluorescent Nanodiamonds as Free Radical Sensors in Aging Yeast Cells: a baker’s yeast response to small diamonds with great potential!. Rijksuniversiteit Groningen.

https://doi.org/10.33612/diss.112906297

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Chapter 7

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General Discussion

Kiran J. van der Laan

The potential benefits of the use of fluorescent nanodiamonds (FNDs) as free radical sensors are enormous, as free radicals are involved in nearly every disease or pathogenic condition. Their exact role remains hard to study since there are currently no precise free radical measurements possible. So there is a big gap too gain here. The methods that are available until now, have some disadvantages that the FNDs can overcome. First of all, there are ways to indirectly measure the activity or presence of free radicals. In biological samples for example by measuring the response of the cells to free radicals, instead of measuring the free radicals directly. This can be done by detecting the activity of the free radical defense system, anti-oxidants, and detection of expression levels of genes that are involved in this oxidative stress response. In addition, there are also some fluorescent markers commercially available. Again these are often indirect measurements, as they mostly depend on the conversion of a metabolite into a fluorescent product. This also gives an insecurity about what exactly is measured, as discussed before, considering that free radicals are highly reactive and might react with dye components or intermediates. Nevertheless, these fluorescent assays, such as the DCFDA assay, are the golden standard at the moment when it is important where the free radicals are built. This is also the reason that this DCFDA assay is being used in our group, to have a reference method to compare the FND magnetometry results with in the future. Furthermore, as these fluorescence colorimetric assays are based on irreversible changes they can only measure an increase in free radicals. This also involves that they are measuring static end points of free radical activity. Using FNDs, both increases and decreases in free radical activity can be measured and one can even record free radical activity during live cell imaging over longer times (limited only by the lifetime of the cell). Additionally, the disadvantage of fluorescent markers and assays is that their fluorescence bleaches. This prevents the possibility of long-term measurements as their signal loses accuracy over time and disappears eventually. As the sensing mechanism is embedded inside the stable FNDs, bleaching is not an issue when using diamond magnetometry. In order to work around the photobleaching issue, available methods could asses the free radical activity over a population of cells so as to compare e.g. a young population with another old population. Whereas with FNDs, you are able to follow the same cell over time in single cell measurements. This allows before and after comparisons within one cell which would be highly valuable because of natural heterogeneity that exists in cell populations. In short, these are the

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General Discussion

Kiran J. van der Laan

The potential benefits of the use of fluorescent nanodiamonds (FNDs) as free radical sensors are enormous, as free radicals are involved in nearly every disease or pathogenic condition. Their exact role remains hard to study since there are currently no precise free radical measurements possible. So there is a big gap too gain here. The methods that are available until now, have some disadvantages that the FNDs can overcome. First of all, there are ways to indirectly measure the activity or presence of free radicals. In biological samples for example by measuring the response of the cells to free radicals, instead of measuring the free radicals directly. This can be done by detecting the activity of the free radical defense system, anti-oxidants, and detection of expression levels of genes that are involved in this oxidative stress response. In addition, there are also some fluorescent markers commercially available. Again these are often indirect measurements, as they mostly depend on the conversion of a metabolite into a fluorescent product. This also gives an insecurity about what exactly is measured, as discussed before, considering that free radicals are highly reactive and might react with dye components or intermediates. Nevertheless, these fluorescent assays, such as the DCFDA assay, are the golden standard at the moment when it is important where the free radicals are built. This is also the reason that this DCFDA assay is being used in our group, to have a reference method to compare the FND magnetometry results with in the future. Furthermore, as these fluorescence colorimetric assays are based on irreversible changes they can only measure an increase in free radicals. This also involves that they are measuring static end points of free radical activity. Using FNDs, both increases and decreases in free radical activity can be measured and one can even record free radical activity during live cell imaging over longer times (limited only by the lifetime of the cell). Additionally, the disadvantage of fluorescent markers and assays is that their fluorescence bleaches. This prevents the possibility of long-term measurements as their signal loses accuracy over time and disappears eventually. As the sensing mechanism is embedded inside the stable FNDs, bleaching is not an issue when using diamond magnetometry. In order to work around the photobleaching issue, available methods could asses the free radical activity over a population of cells so as to compare e.g. a young population with another old population. Whereas with FNDs, you are able to follow the same cell over time in single cell measurements. This allows before and after comparisons within one cell which would be highly valuable because of natural heterogeneity that exists in cell populations. In short, these are the

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GENERAL DISCUSSION

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most important limitations and shortcomings of presently available free radical detection methods. More than that, they highlight the advantages that FNDs can bring for free radical research, including research for the exact role of free radicals in aging.

Aging

In the research presented in this thesis, the role of free radicals in aging was investigated using the well-known and preferred aging model Saccharomyces cerevisiae. The first step here was to introduce the FNDs in yeast cells, in order to minimize the distance between the FND sensor and its quantity of interest, free radicals. In Chapter 2 we successfully developed a method to obtain FND

internalization into yeast cells. We adopted a transformation method to temporarily disrupt the cell membrane to allow the FNDs to move inside. Additionally, we devised a method to quantify the actual uptake of diamonds, using imaging software and microscopic figures, and we confirmed that the cells were still proliferating after being subjected to both the transformation and the FNDs.

After successfully bringing the diamonds inside the cells, we aimed to investigate the effect of our diamonds to the ageing process of the yeast cells in

Chapter 3. We checked for changes in the ageing curves of yeast cells, after

treating them with both the uptake protocol and the diamonds themselves. We found no significant differences in survival at any time point in the first 14 days after diamond internalization. Also, since we found that the diamond uptake was transient and mostly gone again after 24 hours, we investigated another strategy to have diamonds in aged cells. Instead of first adding the diamonds and thereafter ageing the cells, we tried to age the cells first and subsequently add the diamonds at the desired age. We found that in this way the uptake protocol was still effective, and also the aged cells still survived the treatment. Finally, we studied the destination of the diamonds in the cells and found that they were preferentially present in the near vicinity of membrane-enclosed organelles.

Biocompatibility

Biocompatibility refers to the ability of a model organism to react to nonliving material, in this case a yeast cell responding to nanodiamonds, and can be measured in many different ways. Often people point to the cell’s viability in response to a treatment, but cellular viability has many faces. Its definition, the capability to live successfully, already indicates its multidimensional character. Many factors are involved in living successfully and thus many quantities can be measured as of to determine a cell’s viability. Examples of viability measures are proliferative capacity, membrane integrity, metabolic activity and oxidative stress response activation. The latter is specifically interesting with the goal of measuring free radicals in mind, as we want to make sure that we are measuring free radicals in response to our stressor or condition, and not the response to our FNDs. In this thesis, different aspects of the yeast cell viability in response to FNDs were evaluated. The effect of FNDs on proliferation and metabolic activity of yeast cells were discussed before (Chapter 2 and 3). These aspects

can be considered as rough measurements of the cell’s viability, as they concern the survival of the cells. While not seeing an effect on survival rates, there might still be non-fatal changes going on inside the cells. The oxidative stress response of yeast cells to FNDs was evaluated in Chapter 4. Here we found that there was

no change in the general metabolic activity of the cells after adding the FNDs. Likewise there were only minimal FND-related oxidative stress effects detected, confirming the excellent biocompatibility. This knowledge is crucial to make the step towards the actual measurement of free radicals in yeast cells using FNDs.

In Chapter 5 the applications of nanodiamonds in all kinds of cells was

discussed, such as temperature detection and gene delivery. Part of the discussion of these applications included the biocompatibility of different nanodiamonds in the different cell types. For detonation nanodiamonds, cytotoxic effects were only observed after exposure of cells to DNDs at excessive concentrations of over 100 ug/ml, in a concentration-dependent manner. However, the cytotoxic effect showed to not only be dependent on the concentration but also on the type of biological materials (aka cell type). Similar findings were observed for fluorescent nanodiamonds, with no or low cytotoxic effects at reasonable concentrations. But some cytotoxic effects appeared in concentration dependent conditions. Again, this effect also depended on the tested cell line.

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most important limitations and shortcomings of presently available free radical detection methods. More than that, they highlight the advantages that FNDs can bring for free radical research, including research for the exact role of free radicals in aging.

Aging

In the research presented in this thesis, the role of free radicals in aging was investigated using the well-known and preferred aging model Saccharomyces cerevisiae. The first step here was to introduce the FNDs in yeast cells, in order to minimize the distance between the FND sensor and its quantity of interest, free radicals. In Chapter 2 we successfully developed a method to obtain FND

internalization into yeast cells. We adopted a transformation method to temporarily disrupt the cell membrane to allow the FNDs to move inside. Additionally, we devised a method to quantify the actual uptake of diamonds, using imaging software and microscopic figures, and we confirmed that the cells were still proliferating after being subjected to both the transformation and the FNDs.

After successfully bringing the diamonds inside the cells, we aimed to investigate the effect of our diamonds to the ageing process of the yeast cells in

Chapter 3. We checked for changes in the ageing curves of yeast cells, after

treating them with both the uptake protocol and the diamonds themselves. We found no significant differences in survival at any time point in the first 14 days after diamond internalization. Also, since we found that the diamond uptake was transient and mostly gone again after 24 hours, we investigated another strategy to have diamonds in aged cells. Instead of first adding the diamonds and thereafter ageing the cells, we tried to age the cells first and subsequently add the diamonds at the desired age. We found that in this way the uptake protocol was still effective, and also the aged cells still survived the treatment. Finally, we studied the destination of the diamonds in the cells and found that they were preferentially present in the near vicinity of membrane-enclosed organelles.

Biocompatibility

Biocompatibility refers to the ability of a model organism to react to nonliving material, in this case a yeast cell responding to nanodiamonds, and can be measured in many different ways. Often people point to the cell’s viability in response to a treatment, but cellular viability has many faces. Its definition, the capability to live successfully, already indicates its multidimensional character. Many factors are involved in living successfully and thus many quantities can be measured as of to determine a cell’s viability. Examples of viability measures are proliferative capacity, membrane integrity, metabolic activity and oxidative stress response activation. The latter is specifically interesting with the goal of measuring free radicals in mind, as we want to make sure that we are measuring free radicals in response to our stressor or condition, and not the response to our FNDs. In this thesis, different aspects of the yeast cell viability in response to FNDs were evaluated. The effect of FNDs on proliferation and metabolic activity of yeast cells were discussed before (Chapter 2 and 3). These aspects

can be considered as rough measurements of the cell’s viability, as they concern the survival of the cells. While not seeing an effect on survival rates, there might still be non-fatal changes going on inside the cells. The oxidative stress response of yeast cells to FNDs was evaluated in Chapter 4. Here we found that there was

no change in the general metabolic activity of the cells after adding the FNDs. Likewise there were only minimal FND-related oxidative stress effects detected, confirming the excellent biocompatibility. This knowledge is crucial to make the step towards the actual measurement of free radicals in yeast cells using FNDs.

In Chapter 5 the applications of nanodiamonds in all kinds of cells was

discussed, such as temperature detection and gene delivery. Part of the discussion of these applications included the biocompatibility of different nanodiamonds in the different cell types. For detonation nanodiamonds, cytotoxic effects were only observed after exposure of cells to DNDs at excessive concentrations of over 100 ug/ml, in a concentration-dependent manner. However, the cytotoxic effect showed to not only be dependent on the concentration but also on the type of biological materials (aka cell type). Similar findings were observed for fluorescent nanodiamonds, with no or low cytotoxic effects at reasonable concentrations. But some cytotoxic effects appeared in concentration dependent conditions. Again, this effect also depended on the tested cell line.

200 201

GENERAL DISCUSSION

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To study the biocompatibility of organisms with nanodiamonds, one can study the behavior of diamonds inside a body. Chapter 6 focused on the in vivo

applications of nanodiamonds, such as drug delivery and labeling, and also the effect of diamonds on animals was discussed. Here we concluded that nanodiamonds are well-tolerated, summarizing the studies that report on mortality rates, morphological and embryonic changes, biochemical parameters and biodistribution and accumulation data. This opens the way toward clinical application of nanodiamonds.

Together with all the advantages mentioned before, the lack of severe cytotoxic effects combined with the tolerance of model organisms gives room for the use of fluorescent nanodiamonds in biomedical research. This could potentially help the study for the many different pathogenic conditions that are influenced by free radicals.

Future directions

Despite all promising properties and advantages of FNDs, there is still work to be done to get this application in operation. One of the opportunities for enhancement is to further develop the home-built confocal microscope for diamond magnetometry in order to improve sensitivity and time resolution. By introducing new pulsing sequences for t1 and t2 measurements, the sensitivity can be increased which also means that less repetitions are needed to get the desired results which in turn also results in less time needed for the measurement. There are also some challenges left in data interpretation in order to validate and analyze the signals that are obtained. Proof of principle experiments are currently done using the set-up, by comparing to conventional methods, and will soon be presented. Increasing the sensitivity further would be desirable to be able to differentiate between species of free radicals.

Next to making progress in the technical development, there are also some biological challenges still open for further exploring. For example, being able to target diamonds to a specific directions. This is currently attempted by attaching the diamond with antibodies. Being able to send the diamonds to specific subcellular locations, will enable measuring the free radical activity at specific organelles in the cell adding spatial information to the measurements.

Once these challenges are tackled, the motives for collaborations are numerous for every department in the hospital. Moreover, this is not only

interesting for studying the role of free radicals in biomedical conditions. Other possible applications and fields where this technique might be valuable, are discussed in the next paragraph.

Valorisation

In this thesis, the functionality of free radical biosensor is discussed as the main application of FNDs with the goal of providing fundamental knowledge, in the shape of a map with the origin and migration patterns of free radicals. However, there is a broader area of interest that could potentially benefit from diamond magnetometry measurements. In this final section, some of the potential users and fields will be discussed.

Monitoring drug efficacy

Still in the biomedical field: monitoring drug efficacy might be another need where diamond magnetometry with FNDs could be applied. Actually in the past year a start-up company, Diamond Sense, has been founded by several of our group members with Thamir Hamoh as the CEO. The idea of this company is to commercially provide services for parties that are interested in free radical quantification. Customers who are interested in these kind of data, could hand their samples to the company in order to have magnetometry experiments conducted. On the long run, the company is also considering the option to sell chips dedicated for certain tests or to sell whole magnetometry instruments. The company’s aim is to use diamond magnetometry as a high resolution tool to monitor drug efficiency, which could be interesting for pharmaceutical companies, clinical research organization or research groups that work on drug development.

In practice, drug tests using diamond magnetometry could for example involve measuring the response of a cell to the drug. In a stress response, the cell might be going into apoptosis or might start creating free radicals. Potential drugs that could cause these effects are anti-cancer drugs or antibiotics. Another class of drugs, so-called anti-aging drugs, reduce free radical generation rather than increasing. These drugs are particularly interesting for this application, as a decrease is typically more difficult to measure for state of the art dyes (as discussed before).

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To study the biocompatibility of organisms with nanodiamonds, one can study the behavior of diamonds inside a body. Chapter 6 focused on the in vivo

applications of nanodiamonds, such as drug delivery and labeling, and also the effect of diamonds on animals was discussed. Here we concluded that nanodiamonds are well-tolerated, summarizing the studies that report on mortality rates, morphological and embryonic changes, biochemical parameters and biodistribution and accumulation data. This opens the way toward clinical application of nanodiamonds.

Together with all the advantages mentioned before, the lack of severe cytotoxic effects combined with the tolerance of model organisms gives room for the use of fluorescent nanodiamonds in biomedical research. This could potentially help the study for the many different pathogenic conditions that are influenced by free radicals.

Future directions

Despite all promising properties and advantages of FNDs, there is still work to be done to get this application in operation. One of the opportunities for enhancement is to further develop the home-built confocal microscope for diamond magnetometry in order to improve sensitivity and time resolution. By introducing new pulsing sequences for t1 and t2 measurements, the sensitivity can be increased which also means that less repetitions are needed to get the desired results which in turn also results in less time needed for the measurement. There are also some challenges left in data interpretation in order to validate and analyze the signals that are obtained. Proof of principle experiments are currently done using the set-up, by comparing to conventional methods, and will soon be presented. Increasing the sensitivity further would be desirable to be able to differentiate between species of free radicals.

Next to making progress in the technical development, there are also some biological challenges still open for further exploring. For example, being able to target diamonds to a specific directions. This is currently attempted by attaching the diamond with antibodies. Being able to send the diamonds to specific subcellular locations, will enable measuring the free radical activity at specific organelles in the cell adding spatial information to the measurements.

Once these challenges are tackled, the motives for collaborations are numerous for every department in the hospital. Moreover, this is not only

interesting for studying the role of free radicals in biomedical conditions. Other possible applications and fields where this technique might be valuable, are discussed in the next paragraph.

Valorisation

In this thesis, the functionality of free radical biosensor is discussed as the main application of FNDs with the goal of providing fundamental knowledge, in the shape of a map with the origin and migration patterns of free radicals. However, there is a broader area of interest that could potentially benefit from diamond magnetometry measurements. In this final section, some of the potential users and fields will be discussed.

Monitoring drug efficacy

Still in the biomedical field: monitoring drug efficacy might be another need where diamond magnetometry with FNDs could be applied. Actually in the past year a start-up company, Diamond Sense, has been founded by several of our group members with Thamir Hamoh as the CEO. The idea of this company is to commercially provide services for parties that are interested in free radical quantification. Customers who are interested in these kind of data, could hand their samples to the company in order to have magnetometry experiments conducted. On the long run, the company is also considering the option to sell chips dedicated for certain tests or to sell whole magnetometry instruments. The company’s aim is to use diamond magnetometry as a high resolution tool to monitor drug efficiency, which could be interesting for pharmaceutical companies, clinical research organization or research groups that work on drug development.

In practice, drug tests using diamond magnetometry could for example involve measuring the response of a cell to the drug. In a stress response, the cell might be going into apoptosis or might start creating free radicals. Potential drugs that could cause these effects are anti-cancer drugs or antibiotics. Another class of drugs, so-called anti-aging drugs, reduce free radical generation rather than increasing. These drugs are particularly interesting for this application, as a decrease is typically more difficult to measure for state of the art dyes (as discussed before).

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GENERAL DISCUSSION

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Monitoring diagnostics

Next to monitoring drug effects, there might be another opportunity for FNDs in the medical world. Since free radicals play a key role in many diseases, they could also possibly serve as disease indicators in diagnostics. Bacterial infections, cancer, cardiovascular diseases, arthritis and virus infections are some of the diseases which are known to be associated with free radicals. For this direction, it would be wise to connect with the corresponding hospital departments and work together with clinicians. The group already has ongoing collaborations within the UMCG with the Cancer surgery, the Medical Microbiology and the Rheumatology & Clinical Immunology department, and as well with the European Research Institute for the Biology of Ageing (ERIBA).

Monitoring chemical reactions

Outside the medical world, another potential use could be to follow chemical reactions during the fabrication processes of chemicals. This could be interesting for any player in the chemical fabrication field that is in need of optimization of the process while using small available sample sizes. Magnetometry could be applied here to monitor the progress of the reaction by detecting changes in their coherence times. Because of the diamond’s magneto-optical properties, differentiations between reaction mechanisms can be made as well as comparisons between conditions. All together this information can contribute to a more efficient synthesis.

In this context it would not only be interesting to look at the free radical activity, but to utilize other possibilities of the NV centers/FNDs as well. For example to test other quantities involved in chemical reactions such as temperature and pH. This could be interesting for any field that involves fabrication of chemicals, such as organic syntheses, food production and materials sciences, of both companies and research groups. In materials science, a potentially interesting area could be material degradation. If the degradation leads to a radical, this could be measured directly. Another option could be to incorporate diamonds into the material of interest and then measure how it is exposed.

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Monitoring diagnostics

Next to monitoring drug effects, there might be another opportunity for FNDs in the medical world. Since free radicals play a key role in many diseases, they could also possibly serve as disease indicators in diagnostics. Bacterial infections, cancer, cardiovascular diseases, arthritis and virus infections are some of the diseases which are known to be associated with free radicals. For this direction, it would be wise to connect with the corresponding hospital departments and work together with clinicians. The group already has ongoing collaborations within the UMCG with the Cancer surgery, the Medical Microbiology and the Rheumatology & Clinical Immunology department, and as well with the European Research Institute for the Biology of Ageing (ERIBA).

Monitoring chemical reactions

Outside the medical world, another potential use could be to follow chemical reactions during the fabrication processes of chemicals. This could be interesting for any player in the chemical fabrication field that is in need of optimization of the process while using small available sample sizes. Magnetometry could be applied here to monitor the progress of the reaction by detecting changes in their coherence times. Because of the diamond’s magneto-optical properties, differentiations between reaction mechanisms can be made as well as comparisons between conditions. All together this information can contribute to a more efficient synthesis.

In this context it would not only be interesting to look at the free radical activity, but to utilize other possibilities of the NV centers/FNDs as well. For example to test other quantities involved in chemical reactions such as temperature and pH. This could be interesting for any field that involves fabrication of chemicals, such as organic syntheses, food production and materials sciences, of both companies and research groups. In materials science, a potentially interesting area could be material degradation. If the degradation leads to a radical, this could be measured directly. Another option could be to incorporate diamonds into the material of interest and then measure how it is exposed.

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GENERAL DISCUSSION

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