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Introduction: As nanodiamonds become more and more widely used for intracellular labelling and measurements, the task of delivering these nanoparticles inside cells becomes more and more important. Certain cell types easily take up nanodiamonds, while others require special procedures. Methods: In previous research, we found that HT-29 cells (a colon cancer cell line), which are notoriously difficult in the context of nanodiamond internalization, show increased uptake rates, when pre-treated with trypsin- ethylenediaminetetraacetic acid (trypsin-EDTA). However, the uptake mechanism has not been studied before. This article focuses on a more detailed investigation of the reasons underlying this phenomenon. We start by identifying the timing of fluorescent nanodiamond (FND) uptake in trypsin-EDTA pre-treated cells. We then use a combination of chemical inhibitors and Immunocytochemistry to identify the main pathways employed by HT-29 cells in the internalization process. Results and Discussion: We investigate how these pathways are affected by the trypsin-EDTA pre-treatment and conclude by offering possible explanations for this phenomenon. We found that nanodiamonds are internalized via different pathways. Clathrin-mediated endocytosis proves to be the dominating mechanism. Trypsin-EDTA treatment increases particle uptake and affects the uptake mechanism.
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Cardiovascular diseases are currently the most common cause of death in developed countries. Due to lifestyle and environmental factors, this problem is only expected to increase in the future. Reactive oxygen species (ROS) are a key player in the onset of cardiovascular diseases but also have important functions in healthy cardiac tissue. Here, the interplay between ROS generation and cardiac mechanical forces is shown, and the state of the art and a perspective on future directions are discussed. To this end, an overview of what is currently known regarding ROS and mechanosignaling at a subcellular level is first given. There the role of ROS in mechanosignaling as well as the interplay between both factors in specific organelles is emphasized. The consequences at a larger scale across the population of heart cells are then discussed. Subsequently, the roles of ROS in embryogenesis, pathogenesis, and aging are further discussed, exemplifying some aspects of mechanoregulation. Finally, different models that are currently in use are discussed to study the topics above.
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Metals are widely utilized as implant materials for bone fixtures as well as stents. Biodegradable versions of these implants are highly desirable since patients do not have to undergo a second surgery for the materials to be removed. Attractive options for such materials are zinc silver alloys since they also offer the benefit of being antibacterial. However, it is important to investigate the effect of the degradation products of such alloys on the surrounding cells, taking into account silver cytotoxicity. Here we investigated zinc alloyed with 1 % of silver (Zn1Ag) and how differently concentrated extracts (1 %-100 %) of this material impact human umbilical vein endothelial cells (HUVECs). More specifically, we focused on free radical generation and oxidative stress as well as the impact on cell viability. To determine free radical production we used diamond-based quantum sensing as well as conventional fluorescent assays. The viability was assessed by observing cell morphology and the metabolic activity via the MTT assay. We found that 1 % and 10 % extracts are well tolerated by the cells. However, at higher extract concentrations we observed severe impact on cell viability and oxidative stress. We were also able to show that quantum sensing was able to detect significant free radical generation even at the lowest tested concentrations.
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Aleaciones , Supervivencia Celular , Células Endoteliales de la Vena Umbilical Humana , Nanodiamantes , Estrés Oxidativo , Zinc , Humanos , Aleaciones/química , Supervivencia Celular/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Nanodiamantes/química , Plata/toxicidad , Plata/química , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Radicales Libres/metabolismo , Ensayo de Materiales/métodos , Implantes Absorbibles/efectos adversosRESUMEN
Multifunctional wound dressings, enriched with biologically active agents for preventing or treating infections and promoting wound healing, along with cell delivery capability, are highly needed. To address this issue, composite scaffolds with potential in wound dressing applications were fabricated in this study. The poly-lactic acid/nanodiamonds (PLA/ND) scaffolds were first printed using melt electrowriting (MEW) and then coated with quaternized ß-chitin (QßC). The NDs were well-dispersed in the printed filaments and worked as fillers and bioactive additions to PLA material. Additionally, they improved coating effectiveness due to the interaction between their negative charges (from NDs) and positive charges (from QßC). NDs not only increased the thermal stability of PLA but also benefitted cellular behavior and inhibited the growth of bacteria. Scaffolds coated with QßC increased the effect of bacteria growth inhibition and facilitated the proliferation of human dermal fibroblasts. Additionally, we have observed rapid extracellular matrix (ECM) remodeling on QßC-coated PLA/NDs scaffolds. The scaffolds provided support for cell adhesion and could serve as a valuable tool for delivering cells to chronic wound sites. The proposed PLA/ND scaffold coated with QßC holds great potential for achieving fast healing in various types of wounds.
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Ultraviolet (UV) radiation is known to cause skin issues, such as dryness, aging, and even cancer. Among UV rays, UVB stands out for its ability to trigger problems within cells, including mitochondrial dysfunction, oxidative stress, and DNA damage. Free radicals are implicated in these cellular responses, but they are challenging to measure due to their short lifetime and limited diffusion range. In our study, we used a quantum sensing technique (T1 relaxometry) involving fluorescent nanodiamonds (FNDs) that change their optical properties in response to magnetic noise. This allowed us to monitor the free radical presence in real time. To measure radicals near mitochondria, we coated FNDs with antibodies, targeting mitochondrial protein voltage-dependent anion channel 2 (anti-VDAC2). Our findings revealed a dynamic rise in radical levels on the mitochondrial membrane as cells were exposed to UVB (3 J/cm2), with a significant increase observed after 17 min.
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Queratinocitos , Mitocondrias , Rayos Ultravioleta , Humanos , Mitocondrias/metabolismo , Mitocondrias/efectos de la radiación , Radicales Libres/química , Queratinocitos/efectos de la radiación , Queratinocitos/metabolismo , Puntos Cuánticos/química , Puntos Cuánticos/efectos de la radiaciónRESUMEN
Cells are damaged during hypoxia (blood supply deprivation) and reoxygenation (oxygen return). This damage occurs in conditions such as cardiovascular diseases, cancer, and organ transplantation, potentially harming the tissue and organs. The role of free radicals in cellular metabolic reprogramming under hypoxia is under debate, but their measurement is challenging due to their short lifespan and limited diffusion range. In this study, we employed a quantum sensing technique to measure the real-time production of free radicals at the subcellular level. We utilize fluorescent nanodiamonds (FNDs) that exhibit changes in their optical properties based on the surrounding magnetic noise. This way, we were able to detect the presence of free radicals. To specifically monitor radical generation near mitochondria, we coated the FNDs with an antibody targeting voltage-dependent anion channel 2 (anti-VDAC2), which is located in the outer membrane of mitochondria. We observed a significant increase in the radical load on the mitochondrial membrane when cells were exposed to hypoxia. Subsequently, during reoxygenation, the levels of radicals gradually decreased back to the normoxia state. Overall, by applying a quantum sensing technique, the connections among hypoxia, free radicals, and the cellular redox status has been revealed.
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Hipoxia , Miocitos Cardíacos , Humanos , Miocitos Cardíacos/metabolismo , Radicales Libres/metabolismo , Hipoxia/metabolismo , Mitocondrias/metabolismo , Oxígeno/metabolismoRESUMEN
Cumulus granulosa cells (cGCs) and mural granulosa cells (mGCs), although derived from the same precursors, are anatomically and functionally heterogeneous. They are critical for female fertility by supporting oocyte competence and follicular development. There are various techniques used to investigate the role of free radicals in mGCs and cCGs. Yet, temporospatial resolution remains a challenge. We used a quantum sensing approach to study free radical generation at nanoscale in cGCs and mGCs isolated from women undergoing oocyte retrieval during in vitro fertilization (IVF). Cells were incubated with bare fluorescent nanodiamonds (FNDs) or mitochondria targeted FNDs to detect free radicals in the cytoplasm and mitochondria. After inducing oxidative stress with menadione, we continued to detect free radical generation for 30 min. We observed an increase in free radical generation in cGCs and mGCs from 10 min on. Although cytoplasmic and mitochondrial free radical levels are indistinguishable in the physiological state in both cGCs and mGCs, the free radical changes measured in mitochondria were significantly larger in both cell types, suggesting mitochondria are sites of free radical generation. Furthermore, we observed later occurrence and a smaller percentage of cytoplasmic free radical change in cGCs, indicating that cGCs may be more resistant to oxidative stress.
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Diamond-based T1 relaxometry is a new technique that allows nanoscale magnetic resonance measurements. Here we present its first application in patient samples. More specifically, we demonstrate that relaxometry can determine the free radical load in samples from arthritis patients. We found that we can clearly differentiate between osteoarthritis and rheumatoid arthritis patients in both the synovial fluid itself and cells derived from it. Furthermore, we tested how synovial fluid and its cells respond to piroxicam, a common nonsteroidal anti-inflammatory drug (NSAID). It is known that this drug leads to a reduction in reactive oxygen species production in fibroblast-like synoviocytes (FLS). Here, we investigated the formation of free radicals specifically. While FLS from osteoarthritis patients showed a drastic decrease in the free radical load, cells from rheumatoid arthritis retained a similar radical load after treatment. This offers a possible explanation for why piroxicam is more beneficial for patients with osteoarthritis than those with rheumatoid arthritis.
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Artritis Reumatoide , Osteoartritis , Humanos , Líquido Sinovial , Membrana Sinovial/patología , Piroxicam/uso terapéutico , Células Cultivadas , Artritis Reumatoide/diagnóstico por imagen , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/patología , Osteoartritis/diagnóstico por imagen , Osteoartritis/tratamiento farmacológico , Osteoartritis/patología , Fibroblastos/patologíaRESUMEN
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.
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Nanodiamantes , Plásticos , Colorantes Fluorescentes , Sistemas de Liberación de Medicamentos , PolímerosRESUMEN
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.
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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.
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Níquel , Titanio , Especies Reactivas de Oxígeno , Aleaciones/farmacología , Stents , Células Epiteliales , Proliferación Celular , Fibroblastos , PulmónRESUMEN
We present an alternative to conventional Electron Paramagnetic Resonance (EPR) spectroscopy equipment. Avoiding the use of bulky magnets and magnetron equipment, we use the photoluminescence of an ensemble of Nitrogen-Vacancy centers at the surface of a diamond. Monitoring their relaxation time (or T1), we detected their cross-relaxation with a compound of interest. In addition, the EPR spectra are encoded through a localized magnetic field gradient. While recording previous data took 12 min per data point with individual NV centers, we were able to reconstruct a full spectrum at once in 3 s, over a range from 3 to 11 G. In terms of sensitivity, only 0.5 µL of a 1 µM hexaaquacopper(II) ion solution was necessary.
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Diamante , Imanes , Diamante/química , Espectroscopía de Resonancia Magnética/métodos , Espectroscopía de Resonancia por Spin del Electrón/métodos , Campos MagnéticosRESUMEN
A broad spectrum of biomaterials has been explored in order to design cardiovascular implants of sufficient hemocompatibility. Most of them were extensively tested for the ability to facilitate repopulation by patient cells. It was shown that stiffness, surface roughness, or hydrophilicity of polyelectrolyte films have an impact on adhesion, proliferation, and differentiation of cells. At the same time, it is still unknown how these properties influence cell functionality and as a consequence interactions with blood components under dynamic conditions. In this study, we aimed to determine the impact of chemical cross-linking of Chitosan (Chi) and Chrondroitin Sulphate (CS) on endothelium-blood cross-talk. We have found that the morphology of the endothelium monolayer was not altered by changes in coating properties. However, free radical generation by endothelial cells varied depending on the elastic properties of the coating. Simultaneously, we have observed a significant decrease in the level of adhering and circulating active platelets as well as aggregates when the endothelium monolayer was formed on stiffer films than on the other coating variants. Moreover, the same type of films has promoted significantly higher adhesion of blood morphotic elements when they were not functionalized by endothelium. The observed changes in hemocompatibility indicate the importance of a design of coatings that will promote cellularization in vivo in a relatively short time and which will regulate cell function.
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Materiales Biocompatibles , Células Endoteliales , Humanos , Polielectrolitos , Adhesión Celular , Endotelio , Materiales Biocompatibles/farmacología , Materiales Biocompatibles/química , Propiedades de SuperficieRESUMEN
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.
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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.
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Diamante , Nanodiamantes , Diamante/química , Nitrógeno/química , Nanodiamantes/química , Fluorescencia , TemperaturaRESUMEN
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.
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Acetaminophen overdoses cause cell injury in the liver. It is widely accepted that liver toxicity is initiated by the reactive N-acetyl-para-aminophenol (APAP) metabolite N-acetyl-p-benzoquinone imine (NAPQI), which first depletes glutathione and then irreversibly binds to mitochondrial proteins and nuclear DNA. As a consequence, mitochondrial respiration is inhibited, and DNA strands break. NAPQI also promotes the oxidative stress since glutathione is one of the main free-radical scavengers in the cell. However, so far it is unknown where exactly free radicals are generated. In this study, we used relaxometry, a novel technique that allows nanoscale magnetic resonance imaging detection of free radicals. The method is based on fluorescent nanodiamonds, which change their optical properties based on their magnetic surrounding. To achieve subcellular resolution, these nanodiamonds were targeted to cellular locations, that is, the cytoplasm, mitochondria, and the nucleus. Since relaxometry is sensitive to spin noise from radicals, we were able to measure the radical load in these different organelles. For the first time, we measured APAP-induced free-radical production in an organelle-specific manner, which helps predict and better understand cellular toxicity.
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Acetaminofén , Nanodiamantes , Acetaminofén/toxicidad , Citosol/metabolismo , Glutatión , Mitocondrias/metabolismo , Radicales Libres/metabolismo , MacrófagosRESUMEN
Nanotechnology holds great promise and is hyped by many as the next industrial evolution. Medicine, food and cosmetics, agriculture and environmental health, and technology industries already profit from nanotechnology innovations and their influence is expected to increase drastically in the near future. However, there are also many challenges that need to be overcome to bring a nanotechnological product or business to the market. In this article we discuss current examples of nanotechnology that have been successfully introduced in the market and their relevance and geographical spread. We then discuss different partners for scientists and their role in the commercialization process. Finally, we review the different steps it takes to bring a nanotechnology to the market, highlight the many difficulties related to these steps, and provide a roadmap for the journey from lab to industry which can be beneficial to researchers.
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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.
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Diamante , Óxido Nítrico , Nitrógeno , Magnetismo/métodos , MagnetometríaRESUMEN
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.