Nanodiamonds in Advancing Biomedical Sciences.

Facile preparation of fluorescent nanodiamond based polymer nanoparticles via ring-opening polymerization and their biological imaging.

Nanodiamonds (ND), especially fluorescent NDs, represent potentially applicable drug and probe carriers for in vitro/in vivo applications. The main purpose of this study was to relate physical-chemical properties of carboxylated NDs to their intracellular distribution and impact on membranes and cell immunity-activation of inflammasome in the in vitro THP-1 cell line model. Dynamic light scattering, nanoparticle tracking analysis, and microscopic methods were used to characterize ND particles and their intracellular distribution. Fluorescent NDs penetrated the cell membranes by both macropinocytosis and mechanical cutting through cell membranes. We proved accumulation of fluorescent NDs in lysosomes. In this case, lysosomes were destabilized and cathepsin B was released into the cytoplasm and triggered pathways leading to activation of inflammasome NLRP3, as detected in THP-1 cells. Activation of inflammasome by NDs represents an important event that could underlie the described toxicological effects in vivo induced by NDs. According to our knowledge, this is the first in vitro study demonstrating direct activation of inflammasome by NDs. These findings are important for understanding the mechanism(s) of action of ND complexes and explain the ambiguity of the existing toxicological data.

Nanodiamond (ND) has emerged as a promising carbon nanomaterial for therapeutic applications. In previous studies, ND has been reported to have outstanding biocompatibility and high uptake rate in various cell types. ND containing nitrogen-vacancy centers exhibit fluorescence property is called fluorescent nanodiamond (FND), and has been applied for bio-labeling agent. However, the influence and application of FND on the nervous system remain elusive. In order to study the compatibility of FND on the nervous system, neurons treated with FNDs in vitro and in vivo were examined. FND did not induce cytotoxicity in primary neurons from either central (CNS) or peripheral nervous system (PNS); neither did intracranial injection of FND affect animal behavior. The neuronal uptake of FNDs was confirmed using flow cytometry and confocal microscopy. However, FND caused a concentration-dependent decrease in neurite length in both CNS and PNS neurons. Time-lapse live cell imaging showed that the reduction of neurite length was due to the spatial hindrance of FND on advancing axonal growth cone. These findings demonstrate that FNDs exhibit low neuronal toxicity but interfere with neuronal morphogenesis, and should be taken into consideration when applications involve actively growing neurites (e.g. nerve regeneration).

Since nanodiamonds (NDs) exhibit low or no toxicity, NDs have attracted growing interest as promising materials for biomedical applications. Herein are reported size separation of ND particles by use of ultracentrifugation and synthesis of fluorescent ND through the surface chemical modifications. The convenient size separation method by use of ultracentrifugation gave desired sizes of NDs ranging from 4 to 25 nm in medium diameters by controlling the duration and acceleration of the centrifugation. The ND size is important for passive targeting such as EPR effect in their applications for DDS and tumor imaging. Surface chemical modification is also vital for adding requisite functions to NDs such as dispersibility and visibility. Step-wise transformations on ND surface were performed, providing fluorescent ND. The ND powder with 30 nm medium diameter was hydrogenated, followed by functionalization with aminoalkyl group via radical reaction. Fluorescent tag was immobilized onto the ND surface through amide linkage, which was confirmed by fluorescent image of the ND.

Measuring physical quantities in the nanometric region inside single cells is of great importance for understanding cellular activity. Thus, the development of biocompatible, sensitive, and reliable nanobiosensors is essential for progress in biological research. Diamond nanoparticles containing nitrogen-vacancy centers (NVCs), referred to as fluorescent nanodiamonds (FNDs), have recently emerged as the sensors that show great promise for ultrasensitive nanosensing of physical quantities. FNDs emit stable fluorescence without photobleaching. Additionally, their distinctive magneto-optical properties enable an optical readout of the quantum states of the electron spin in NVC under ambient conditions. These properties enable the quantitative sensing of physical parameters (temperature, magnetic field, electric field, pH, etc.) in the vicinity of an FND; hence, FNDs are often described as “quantum sensors”. In this review, recent advancements in biosensing applications of FNDs are summarized. First, the principles of orientation and temperature sensing using FND quantum sensors are explained. Next, we introduce surface coating techniques indispensable for controlling the physicochemical properties of FNDs. The achievements of practical biological sensing using surface-coated FNDs, including orientation, temperature, and thermal conductivity, are then highlighted. Finally, the advantages, challenges, and perspectives of the quantum sensing of FND are discussed. This review article is an extended version of the Japanese article, In Situ Measurement of Intracellular Thermal Conductivity Using Diamond Nanoparticle, published in SEIBUTSU BUTSURI Vol. 62, p. 122–124 (2022).

Single Nitrogen-Vacancy Imaging in Nanodiamonds for Multimodal Sensing

Fluorescent nanodiamonds (NDs) coated with therapeutics and cell-targeting structures serve as effective tools for drug delivery. However, NDs circulating in blood can eventually interact with the blood-brain barrier, resulting in undesired pathology. Here, we aimed to detect interaction between NDs and adult brain tissue. First, we cultured neuronal tissue with ND ex vivo and studied cell prosperity, regeneration, cytokine secretion, and nanodiamond uptake. Then, we applied NDs systemically into C57BL/6 animals and assessed accumulation of nanodiamonds in brain tissue and cytokine response. We found that only non-neuronal cells internalized coated nanodiamonds and responded by excretion of interleukin-6 and interferon-γ. Cells of neuronal origin expressing tubulin beta-III did not internalize any NDs. Once we applied coated NDs intravenously, we found no presence of NDs in the adult cortex but observed transient release of interleukin-1α. We conclude that specialized adult neuronal cells do not internalize plain or coated NDs. However, coated nanodiamonds interact with non-neuronal cells present within the cortex tissue. Moreover, the coated NDs do not cross the blood-brain barrier but they interact with adjacent barrier cells and trigger a temporary cytokine response. This study represents the first report concerning interaction of NDs with adult brain tissue.

All the carbon based nanomaterials (CNMs) are highly hydrophobic, which make them unsuitable for most of the applications in water and organic solvents. Aggregation phenomena significantly reduce the high performances displayed by the single nanostructure. Two main strategies allow to overcome this bottleneck: the chemical functionalization with hydrophilic functional groups or the non-covalent interaction between CNMs and amphiphilic molecules. The aim of this thesis has been to produce different carbon-based hybrid nanostructures to preserve the peculiar properties of CNMs and use them for advanced application in nanomedical and technological fields. In the first project, the potential application of fullerene (C60) as sensitizer for photodynamic therapy was explored. Monodispersity of fullerenes is the key feature for its potential application in this field. Noncovalent approach was used to disperse C60 in water, taking advantage from the surfactant-like properties of the proteins. C60@lyszoyme hybrid was used as model system to study the stability of fullerene in physiological conditions and to assess its ability to produce reactive oxygen species upon irradiations. The second subject of my research concerned the study of interactions between fluorescent nanodiamonds (FNDs) and plasma proteins. FNDs show potential applications as probe for bioimaging but their tendency to aggregate in physiological environments is the main limit for their application. In this study, a procedure to keep monodispersed FNDs in relevant biological fluids was optimized and the composition of FNDs protein corona was extensively characterized. The third project was addressed to the manufacturing of graphene based calcite nanocomposite. Both covalent (graphene oxide) and non-covalent (graphene/biomolecules adducts) approaches were used to disperse graphene in water. Following a biological inspired synthetic procedure, it was possible to incorporate the 2D materials within a 3D crystal lattice, producing a nanocomposite possessing several new properties.

Nanotheranostics, combining diagnostics and therapy, has the potential to revolutionize treatment of neurological disorders. But one of the major obstacles for treating central nervous system diseases is the blood-brain barrier (BBB) preventing systemic delivery of drugs and optical probes into the brain. To overcome these limitations, nanodiamonds (NDs) are investigated in this study as they are a powerful sensing and imaging platform for various biological applications and possess outstanding stable far-red fluorescence, do not photobleach, and are highly biocompatible. Herein, fluorescent NDs encapsulated by a customized human serum albumin-based biopolymer (polyethylene glycol) coating (dcHSA-PEG) are taken up by target brain cells. In vitro BBB models reveal transcytosis and an additional direct cell-cell transport via tunneling nanotubes. Systemic application of dcHSA-NDs confirms their ability to cross the BBB in a mouse model. Tracking of dcHSA-NDs is possible at the single cell level and reveals their uptake into neurons and astrocytes in vivo. This study shows for the first time systemic NDs brain delivery and suggests transport mechanisms across the BBB and direct cell-cell transport. Fluorescent NDs are envisioned as traceable transporters for in vivo brain imaging, sensing, and drug delivery.

The relationship between the unique characteristics of nanodiamonds (NDs) and the fluorescence properties of nitrogen-vacancy (NV) centers has lead to a tool with quantum sensing capabilities and nanometric spatial resolution; this tool is able to operate in a wide range of temperatures and pressures and in harsh chemical conditions. For the development of devices based on NDs, a great effort has been invested in researching cheap and easily scalable synthesis techniques for NDs and NV-NDs. In this review, we discuss the common fluorescent NDs synthesis techniques as well as the laser-assisted production methods. Then, we report recent results regarding the applications of fluorescent NDs, focusing in particular on sensing of the environmental parameters as well as in catalysis. Finally, we underline that the highly non-equilibrium processes occurring in the interactions of laser-materials in controlled laboratory conditions for NDs synthesis present unique opportunities for investigation of the phenomena occurring under extreme thermodynamic conditions in planetary cores or under warm dense matter conditions.

Temperature is anessential parameter in all biological systems,but information about the actual temperature in living cells is limited.Especially, in photothermal therapy, local intracellular temperaturechanges induce cell death but the local temperature gradients arenot known. Highly sensitive nanothermometers would be required tomeasure and report local temperature changes independent of the intracellularenvironment, including pH or ions. Fluorescent nanodiamonds (ND) enabletemperature sensing at the nanoscale independent of external conditions.Herein, we prepare ND nanothermometers coated with a nanogel shelland the photothermal agent indocyanine green serves as a heat generatorand sensor. Upon irradiation, programmed cell death was induced incancer cells with high spatial control. In parallel, the increasein local temperature was recorded by the ND nanothermometers. Thisapproach represents a great step forward to record local temperaturechanges in different cellular environments inside cells and correlatethese with thermal biology.

Influence of surface composition on the colloidal stability of ultra-small detonation nanodiamonds in biological media

Nanodiamond (ND) with nitrogen vacancy (NV-) color centers has emerged as an important material for quantum sensing and imaging. Fluorescent, carboxylated ND (140 nm) is investigated for the detection of dopamine (DA), caffeine (CA), and ascorbic acid (AA). Over a 200 nM range, DA and CA quenched the ND fluorescence by 7.1 and 9.8%, respectively. For AA, fluorescence was quenched (2.9%) at nM concentrations and enhanced at μM concentrations. The quenching fit well to Langmuir adsorption isotherms. For DA-CA mixtures, the CA at nM concentrations did not affect DA quenching but interfered when at μM concentrations. The DA at nM or μM concentrations lessened CA quenching. For DA-AA mixtures with DA at mM concentrations, AA quenched fluorescence throughout the nM and μM range, with increased quenching in the nM range. These studies support ND fluorescence modulation as a possible sensor modality for bioanalyte detection.

Due to the large number of possible applications in quantum technology fields—especially regarding quantum sensing—of nitrogen-vacancy (NV) centers in nanodiamonds (NDs), research on a cheap, scalable and effective NDs synthesis technique has acquired an increasing interest. Standard production methods, such as detonation and grinding, require multistep post-synthesis processes and do not allow precise control in the size and fluorescence intensity of NDs. For this reason, a different approach consisting of pulsed laser ablation of carbon precursors has recently been proposed. In this work, we demonstrate the synthesis of NV-fluorescent NDs through pulsed laser ablation of an N-doped graphite target. The obtained NDs are fully characterized in the morphological and optical properties, in particular with optically detected magnetic resonance spectroscopy to unequivocally prove the NV origin of the NDs photoluminescence. Moreover, to compare the different fluorescent NDs laser-ablation-based synthesis techniques recently developed, we report an analysis of the effect of the medium in which laser ablation of graphite is performed. Along with it, thermodynamic aspects of the physical processes occurring during laser irradiation are analyzed. Finally, we show that the use of properly N-doped graphite as a target for laser ablation can lead to precise control in the number of NV centers in the produced NDs.

In recent years, diamond nanoparticles have received a great deal of attention due to their unique photophysical and biological properties. Nanodiamonds (NDs) show low toxicity and are considered to be a highly biocompatible carbon nanomaterial useful in a wide range of applications. Thanks to their ability to accommodate nitrogen-vacancy (N-V) color centers, NDs are a prime example of non-photobleachable fluorescent labels and nanosensors. Here, we present a survey of ND applications in biology and medicine with an emphasis on bio-imaging. We focus on distinguishing the properties of detonation NDs and high-pressure high-temperature (HPHT) NDs and describing their physicochemical properties, structure and possible modifications by small molecules and biomolecules. We summarize and critically evaluate in vitro and in vivo data on ND toxicity and biocompatibility, cellular internalization, localization and targeting by surface-attached ligands. We discuss current achievements in bioimaging using fluorescent NDs and the potential of NDs in diagnostics and drug delivery.