Fluorescent Nanodiamonds for Tracking Single Polymer Particles in Cells and Tissues.

Polymer nanoparticles are widely used in drug delivery and are also a potential concern due to the increased burden of nano- or microplastics in the environment. In order to use polymer nanoparticles safely and understand their mechanism of action, it is useful to know where within cells and tissues they end up. To this end, we labeled polymer nanoparticles with nanodiamond particles. More specifically, we have embedded nanodiamond particles in the polymer particles and characterized the composites. Compared to conventional fluorescent dyes, these labels have the advantage that nanodiamonds do not bleach or blink, thus allowing long-term imaging and tracking of polymer particles. We have demonstrated this principle both in cells and entire liver tissues.

Host-device interactions: exposure of lung epithelial cells and fibroblasts to nickel, titanium, or nitinol affect proliferation, reactive oxygen species production, and cellular signaling.

Endoscopic implantation of medical devices for the treatment of lung diseases, including airway stents, unidirectional valves and coils, is readily used to treat central airway disease and emphysema. However, granulation and fibrotic tissue formation impairs treatment effectiveness. To date little is known about the interaction between implanted devices, often made from metals, such as nickel, titanium or nitinol, and cells in the airways. Here, we study the response of lung epithelial cells and fibroblasts to implant device materials. The adhesion and proliferation of bronchial epithelial cells and lung fibroblasts upon exposure to 10 × 3 × 1 mm pieces of nickel, titanium or nitinol is examined using light and scanning electron microscopy. Pro-inflammatory cytokine mRNA expression and release, signaling kinase activity and intracellular free radical production are assessed. Nitinol, and to a lesser extent nickel and titanium, surfaces support the attachment and growth of lung epithelial cells. Nitinol induces a rapid and significant alteration of kinase activity. Cells directly exposed to nickel or titanium produce free radicals, but those exposed to nitinol do not. The response of lung epithelial cells and fibroblasts depends on the metal type to which they are exposed. Nitinol induces cellular surface growth and the induction of kinase activity, while exposure of lung epithelial cells to nickel and titanium induces free radical production, but nitinol does not.

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.

Diamond Relaxometry as a Tool to Investigate the Free Radical Dialogue between Macrophages and Bacteria.

Although free radicals, which are generated by macrophages play a key role in antimicrobial activities, macrophages sometimes fail to kill Staphylococcus aureus (S. aureus) as bacteria have evolved mechanisms to withstand oxidative stress. In the past decades, several ROS-related staphylococcal proteins and enzymes were characterized to explain the microorganism's antioxidative defense system. Yet, time-resolved and site-specific free radical/ROS detection in bacterial infection were full of challenges. In this work, we utilize diamond-based quantum sensing for studying alterations of the free radical response near S. aureus in macrophages. To achieve this goal we used S. aureus-fluorescent nanodiamond conjugates and measured the spin-lattice relaxation (T1) of NV defects embedded in nanodiamonds. We observed an increase of intracellular free radical generation when macrophages were challenged with S. aureus. However, under a high intracellular oxidative stress environment elicited by lipopolysaccharides, a lower radical load was recorded on the bacteria surfaces. Moreover, by performing T1 measurements on the same particles at different times postinfection, we found that radicals were dominantly scavenged by S. aureus from 80 min postinfection under a high intracellular oxidative stress environment.

Relaxometry with Nitrogen Vacancy (NV) Centers in Diamond.

Relaxometry is a technique which makes use of a specific crystal lattice defect in diamond, the so-called NV center. This defect consists of a nitrogen atom, which replaces a carbon atom in the diamond lattice, and an adjacent vacancy. NV centers allow converting magnetic noise into optical signals, which dramatically increases the sensitivity of the readout, allowing for nanoscale resolution. Analogously to T1 measurements in conventional magnetic resonance imaging (MRI), relaxometry allows the detection of different concentrations of paramagnetic species. However, since relaxometry allows very local measurements, the detected signals are from nanoscale voxels around the NV centers. As a result, it is possible to achieve subcellular resolutions and organelle specific measurements.A relaxometry experiment starts with polarizing the spins of NV centers in the diamond lattice, using a strong laser pulse. Afterward the laser is switched off and the NV centers are allowed to stochastically decay into the equilibrium mix of different magnetic states. The polarized configuration exhibits stronger fluorescence than the equilibrium state, allowing one to optically monitor this transition and determine its rate. This process happens faster at higher levels of magnetic noise. Alternatively, it is possible to conduct T1 relaxation measurements from the dark to the bright equilibrium by applying a microwave pulse which brings NV centers into the -1 state instead of the 0 state. One can record a spectrum of T1 at varying strengths of the applied magnetic field. This technique is called cross-relaxometry. Apart from detecting magnetic signals, responsive coatings can be applied which render T1 sensitive to other parameters as pH, temperature, or electric field. Depending on the application there are three different ways to conduct relaxometry experiments: relaxometry in moving or stationary nanodiamonds, scanning magnetometry, and relaxometry in a stationary bulk diamond with a stationary sample on top.In this Account, we present examples for various relaxometry modes as well as their advantages and limitations. Due to the simplicity and low cost of the approach, relaxometry has been implemented in many different instruments and for a wide range of applications. Herein we review the progress that has been achieved in physics, chemistry, and biology. Many articles in this field have a proof-of-principle character, and the full potential of the technology still waits to be unfolded. With this Account, we would like to stimulate discourse on the future of relaxometry.

Intracellular Relaxometry, Challenges, and Future Directions.

Nitrogen vacancy (NV) centers change their optical properties on the basis of their magnetic surroundings. Since optical signals can be detected more sensitively than small magnetic signals, this technique allows unprecedented sensitivity. Recently, NV center-based relaxometry has been used for measurements in living cells with subcellular resolution. The aim of this Outlook is to identify challenges in the field, including controlling the location of sensing particles, limitations in reproducibility, and issues arising from biocompatibility. We further provide an outlook and point to new directions in the field. These include new diamond materials with NV centers, other defects, or even entirely new materials that might replace diamonds. We further discuss new and more challenging samples, such as tissues or even entire organisms, that might be investigated with NV centers. Then, we address future challenges that have to be resolved in order to achieve this goal. Finally, we discuss new quantities that could be measured with NV centers in the future.

Diamond-Based Nanoscale Quantum Relaxometry for Sensing Free Radical Production in Cells.

Diamond magnetometry makes use of fluorescent defects in diamonds to convert magnetic resonance signals into fluorescence. Because optical photons can be detected much more sensitively, this technique currently holds several sensitivity world records for room temperature magnetic measurements. It is orders of magnitude more sensitive than conventional magnetic resonance imaging (MRI) for detecting magnetic resonances. Here, the use of diamond magnetometry to detect free radical production in single living cells with nanometer resolution is experimentally demonstrated. This measuring system is first optimized and calibrated with chemicals at known concentrations. These measurements serve as benchmarks for future experiments. While conventional MRI typically has millimeter resolution, measurements are performed on individual cells to detect nitric oxide signaling at the nanoscale, within 10-20 nm from the internalized particles localized with a diffraction limited optical resolution. This level of detail is inaccessible to the state-of-the-art techniques. Nitric oxide is detected and the dynamics of its production and inhibition in the intra- and extracellular environment are followed.

Single-Particle Tracking and Trajectory Analysis of Fluorescent Nanodiamonds in Cell-Free Environment and Live Cells.

Diamond magnetometry can provide new insights on the production of free radicals inside live cells due to its high sensitivity and spatial resolution. However, the measurements often lack intracellular context for the recorded signal. In this paper, the possible use of single-particle tracking and trajectory analysis of fluorescent nanodiamonds (FNDs) to bridge that gap is explored. It starts with simulating a set of different possible scenarios of a particle's movement, reflecting different modes of motion, degrees of confinement, as well as shapes and sizes of that confinement. Then, the insights from the analysis of the simulated trajectories are applied to describe the movement of FNDs in glycerol solutions. It is shown that the measurements are in good agreement with the previously reported findings and that trajectory analysis yields meaningful results, when FNDs are tracked in a simple environment. Then the much more complex situation of FNDs moving inside a live cell is focused. The behavior of the particles after different incubation times is analyzed, and the possible intracellular localization of FNDs is deducted from their trajectories. Finally, this approach is combined with long-term magnetometry methods to obtain maps of the spin relaxation dynamics (or T1) in live cells, as FNDs move through the cytosol.

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.

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.

Not all cells are created equal - endosomal escape in fluorescent nanodiamonds in different cells.

Successful delivery of fluorescent nanodiamonds (FNDs) into the cytoplasm is essential to many biological applications. Other applications require FNDs to stay within the endosomes. The diversity of cellular uptake of FNDs and following endosomal escape are less explored. In this article, we quantify particle uptake at a single cell level. We report that FNDs enter into the cells gradually. The number of internalized FNDs per cell differs significantly for the cell lines we investigated at the same incubation time. In HeLa cells we do not see any significant endosomal escape. We also found a wide distribution of FND endosomal escape efficiency within the same cell type. However, compared with HeLa cells, FNDs in HUVECs can easily escape from the endosomes and less than 25% FNDs remained in the vesicles after 4 h incubation time. We believe this work can bring more attention to the diversity of the cells and provide potential guidelines for future studies.

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.

Nanodiamond uptake in colon cancer cells: the influence of direction and trypsin-EDTA treatment.

Nanoparticles are routinely used in cell biology. They deliver drugs or function as labels or sensors. For many of these applications it is essential that the nanoparticles enter the cells. While some cell types readily ingest all kinds of particles, others just don't. We report that uptake can be enhanced for some cells if the particles are administered from the basolateral side of the cells (in this case from below). Compared to apical uptake (from above), we report an 8-fold increase in the number of fluorescent nanodiamonds internalized by the colon cancer cell line HT29. Up to 96% of the cells treated by a modified protocol contain at least one nanodiamond, whereas in the control group we could observe nanodiamonds in less than half of the cells. We were also able to show that simple treatment of cell clusters with trypsin-EDTA leads to the same enhancement of the nanodiamond uptake as seeding the cells on top of the nanoparticles. Although our study is focused on nanodiamonds in HT29 cells, we believe that this method could also be applicable for other nanoparticles and cells with a specific directionality.

Optical Detection of Intracellular Quantities Using Nanoscale Technologies.

Optical probes that can be used to measure certain quantities with subcellular resolution give us access to a new level of information at which physics, chemistry, life sciences, and medicine become strongly intertwined. The emergence of these new technologies is owed to great advances in the physical sciences. However, evaluating and improving these methods to new standards requires a joint effort with life sciences and clinical practice. In this Account, we give an overview of the probes that have been developed for measuring a few highly relevant parameters at the subcellular scale: temperature, pH, oxygen, free radicals, inorganic ions, genetic material, and biomarkers. Luminescent probes are available in many varieties, which can be used for measuring temperature, pH, and oxygen. Since they are influenced by virtually any metabolic process in the healthy or diseased cell, these quantities are extremely useful to understand intracellular processes. Probes for them can roughly be divided into molecular dyes with a parameter dependent fluorescence or phosphorescence and nanoparticle platforms. Nanoparticle probes can provide enhanced photostability, measurement quality, and potential for multiple functionalities. Embedding into coatings can improve biocompatibility or prevent nonspecific interactions between the probe and the cellular environment. These qualities need to be matched however with good uptake properties, colloidal properties and eventually intracellular targeting to optimize their practical applicability. Inorganic ions constitute a broad class of compounds or elements, some of which play specific roles in signaling, while others are toxic. Their detection is often difficult due to the cross-talk with similar ions, as well as other parameters. The detection of free radicals, DNA, and biomarkers at extremely low levels has significant potential for biomedical applications. Their presence is linked more directly to physiological and clinical manifestations. Since existing methods for free radical detection are generally poor in sensitivity and spatiotemporal resolution, new reliable methods that are generally applicable can contribute greatly to advancing this topic in biology. Optical methods that detect DNA or RNA and protein biomarkers exist for intracellular applications, but are mostly relevant for the development of rapid point-of-care sample testing. To elucidate the inner workings of cells, focused multidisciplinary research is required to define the validity and limitations of a nanoparticle probe, in both physical and biological terms. Multifunctional platforms and those that are easily made compatible with conventional research equipment have an edge over other techniques in growing the body of research evidencing their versatility.

Toward Using Fluorescent Nanodiamonds To Study Chronological Aging in Saccharomyces cerevisiae.

One of the theories aiming to explain cellular aging is the free radical theory of aging, which describes the possible role of increased production and accumulation of free radicals. Fluorescent nanodiamonds (FNDs) are proposed to provide a tool to detect these radicals, as they function as magnetic sensors that change their optical properties depending on their magnetic surrounding. Therefore, they could enable the study of aging at a molecular level and unravel the exact role of free radicals in this process. In this study, important steps toward this goal are made. FNDs are introduced in chronologically aging yeast cells. Furthermore, the behavior of FNDs in these aging cells is studied to demonstrate the potency of using FNDs in the search for causes of cellular aging.