Fluorescent nanodiamond labels: Size and concentration matters for sperm cell viability.

Nanodiamonds are increasingly popular in biomedical applications, including optical labelling, drug delivery and nanoscale sensing. Potential new applications are in studying infertility or labelling sperm cells. However, for these applications, it is necessary that nanodiamonds are inert and do not alter sperm properties. In this article, we assessed the biocompatibility of nanodiamonds in detail. We investigated different sizes and concentrations of nanodiamonds and sperm preparation methods. We evaluated if the metabolic activity, membrane integrity, morphology and formation of reactive oxygen species were altered. These parameters were tested for sperm cells in their uncapacitated and capacitated states. Unfortunately, FNDs are not universally biocompatible. Generally, cells in the capacitated state are more prone to stress. Additionally, larger particles and lower concentrations are tolerated better than smaller and higher concentrated particles.

Functionalized Fluorescent Nanodiamonds for Simultaneous Drug Delivery and Quantum Sensing in HeLa Cells.

Here, we present multifunctional fluorescent nanodiamonds (FNDs) for simultaneous drug delivery and free radical detection. For this purpose, we modified FNDs containing nitrogen vacancy (NV) centers with a diazoxide derivative. We found that our particles enter cells more easily and are able to deliver this cancer drug into HeLa cells. The particles were characterized by infrared spectroscopy, dynamic light scattering, and secondary electron microscopy. Compared to the free drug, we observe a sustained release over 72 h rather than 12 h for the free drug. Apart from releasing the drug, with these particles, we can measure the drug's effect on free radical generation directly. This has the advantage that the response is measured locally, where the drug is released. These FNDs change their optical properties based on their magnetic surrounding. More specifically, we make use of a technique called relaxometry to detect spin noise from the free radical at the nanoscale with subcellular resolution. We further compared the results from our new technique with a conventional fluorescence assay for the detection of reactive oxygen species. This provides a new method to investigate the relationship between drug release and the response by the cell via radical formation or inhibition.

Nanoscale MRI for Selective Labeling and Localized Free Radical Measurements in the Acrosomes of Single Sperm Cells.

Free radicals play a major role in sperm development, including maturation and fertilization, but they are also linked to infertility. Since they are short-lived and reactive, they are challenging to detect with state of the art methodologies. Thus, many details surrounding their role remain unknown. One unknown factor is the source of radicals that plays a role in the sperm maturation process. Two alternative sources have been postulated: First, the NADPH-oxidase system embedded in the plasma membrane (NOX5) and second, the NADH-dependent oxidoreductase of mitochondria. Due to a lack of localized measurements, the relative contribution of each source for capacitation remains unknown. To answer this question, we use a technique called diamond magnetometry, which allows nanoscale MRI to perform localized free radical detection. With this tool, we were able to quantify radical formation in the acrosome of sperm heads. This allowed us to quantify radical formation locally in real time during capacitation. We further investigated how different inhibitors or triggers alter the radical generation. We were able to identify NOX5 as the prominent source of radical generation in capacitation while the NADH-dependent oxidoreductase of mitochondria seems to play a smaller role.

Quantum Sensing of Free Radicals in Primary Human Dendritic Cells.

Free radicals are crucial indicators for stress and appear in all kinds of pathogenic conditions, including cancer, cardiovascular diseases, and infection. However, they are difficult to detect due to their reactivity and low abundance. We use relaxometry for the detection of radicals with subcellular resolution. This method is based on a fluorescent defect in a diamond, which changes its optical properties on the basis of the magnetic surroundings. This technique allows nanoscale MRI with unprecedented sensitivity and spatial resolution. Recently, this technique was used inside living cells from a cell line. Cell lines differ in terms of endocytic capability and radical production from primary cells derived from patients. Here we provide the first measurements of phagocytic radical production by the NADPH oxidase (NOX2) in primary dendritic cells from healthy donors. The radical production of these cells differs greatly between donors. We investigated the cell response to stimulation or inhibition.

Fluorescent Nanodiamonds for Detecting Free-Radical Generation in Real Time during Shear Stress in Human Umbilical Vein Endothelial Cells.

Free-radical generation is suspected to play a key role in cardiovascular diseases. Another crucial factor is shear stress. Human umbilical vein endothelial cells (HUVECS), which form the lining of blood vessels, require a physiological shear stress to activate many vasoactive factors. These are needed for maintaining vascular cell functions such as nonthrombogenicity, regulation of blood flow, and vascular tone. Additionally, blood clots form at regions of high shear stress within a blood vessel. Here, we use a new method called diamond magnetometry which allows us to measure the dynamics of free-radical generation in real time under shear stress. This quantum sensing technique allows free-radical detection with nanoscale resolution at the single-cell level. We investigate radical formation in HUVECs in a microfluidic environment under different flow conditions typically found in veins and arteries. Here, we looked into free-radical formation before, during, and after flow. We found that the free-radical production varied depending on the flow conditions. To confirm the magnetometry results and to differentiate between radicals, we performed conventional fluorescent reactive oxygen species (ROS) assays specific for superoxide, nitric oxide, and overall ROS.

Male subfertility and oxidative stress.

To date 15% of couples are suffering from infertility with 45-50% of males being responsible. With an increase in paternal age as well as various environmental and lifestyle factors worsening these figures are expected to increase. As the so-called free radical theory of infertility suggests, free radicals or reactive oxygen species (ROS) play an essential role in this process. However, ROS also fulfill important functions for instance in sperm maturation. The aim of this review article is to discuss the role reactive oxygen species play in male fertility and how these are influenced by lifestyle, age or disease. We will further discuss how these ROS are measured and how they can be avoided during in-vitro fertilization.

Targeting Nanodiamonds to the Nucleus in Yeast Cells.

Nanodiamonds are widely used for drug delivery, labelling or nanoscale sensing. For all these applications it is highly beneficial to have control over the intracellular location of the particles. For the first time, we have achieved targeting the nucleus of yeast cells. In terms of particle uptake, these cells are challenging due to their rigid cell wall. Thus, we used a spheroplasting protocol to remove the cell wall prior to uptake. To achieve nuclear targeting we used nanodiamonds, which were attached to antibodies. When using non-targeted particles, only 20% end up at the nucleus. In comparison, by using diamonds linked to antibodies, 70% of the diamond particles reach the nucleus.

The Fate of Lipid-Coated and Uncoated Fluorescent Nanodiamonds during Cell Division in Yeast.

Fluorescent nanodiamonds are frequently used as biolabels. They have also recently been established for magnetic resonance and temperature sensing at the nanoscale level. To properly use them in cell biology, we first have to understand their intracellular fate. Here, we investigated, for the first time, what happens to diamond particles during and after cell division in yeast (Saccharomyces cerevisiae) cells. More concretely, our goal was to answer the question of whether nanodiamonds remain in the mother cells or end up in the daughter cells. Yeast cells are widely used as a model organism in aging and biotechnology research, and they are particularly interesting because their asymmetric cell division leads to morphologically different mother and daughter cells. Although yeast cells have a mechanism to prevent potentially harmful substances from entering the daughter cells, we found an increased number of diamond particles in daughter cells. Additionally, we found substantial excretion of particles, which has not been reported for mammalian cells. We also investigated what types of movement diamond particles undergo in the cells. Finally, we also compared bare nanodiamonds with lipid-coated diamonds, and there were no significant differences in respect to either movement or intracellular fate.