Nanodiamond Solid-Phase Extraction and Triton X-114 Cloud Point Separation for Robust Fractionation and Shotgun Proteomics Analysis of the Human Serum Proteome.

Human serum is one of the most attractive specimens in biomarker research. However, its overcomplicated properties have hindered the analysis of low-abundance proteins by conventional mass spectrometry techniques. This work proposes an innovative strategy for utilizing nanodiamonds (NDs) in combination with Triton X-114 protein extraction to fractionate the crude serum to six pH-tuned fractions, simplifying the overall proteome and facilitating protein profiling with high efficiency. A total of 663 proteins are identified and evenly distributed among the fractions along with 39 FDA-approved biomarkers─a remarkable increase from the 230 proteins found in unfractionated crude serum. In the low-abundance protein section, 88 proteins with 7 FDA-approved biomarkers are detected─a marked increase from the 15 proteins (2 biomarkers) observed in the untreated sample. Notably, fractions at pH 11, derived from the aqueous phase of detergent separation, suggest potential applications in rapid and robust serum proteome analysis. Notably, by outlining the excellent properties of NDs for proteomic research, this work suggests a promising extraction protocol utilizing the great compatibility of NDs with streamlined serum proteomics and identifies potential avenues for future developments. Finally, we believe that this work not just improves shotgun proteomics but also opens up studies on the interaction between NDs and the human proteome. Data are available via ProteomeXchange with the identifier PXD029710.

Biodistribution of Detonation Synthesis Nanodiamonds in Mice after Intravenous Administration and Some Biochemical Changes in Blood Plasma.

Biodistribution of nanodiamonds in mice after intravenous administration, activities of AST and ALT, and the level of bilirubin in the blood plasma were studied in 2.5 h and 10, 35, and 97 days after injection of nanodiamonds. In 2.5 h after intravenous injection, nanodiamonds mainly accumulate in the lungs and liver. Then, redistribution of nanodiamonds from all organs to the liver was observed. Activities of AST and ALT and the level of bilirubin in the blood increased after 2.5 h and then decreased to the initial values.

Detection of nanodiamonds in biological samples by EPR spectrometry.

In model experiments in vitro, the applicability of the EPR spectrometry method for the detection of modified nanodiamonds (MNDs) in blood and homogenates of mouse organs has been established. A characteristic signal (g = 2.003, ΔH ≈ 10 G) is observed in the samples of biomaterials containing MNDs, the intensity of which increases linearly with the concentration of nanoparticles in the range of 1.6-200 µg MNDs per 1 mL of the sample. The EPR method in biomaterials reveals the presence of intrinsic paramagnetic centers, signals from which are superimposed on the signal from the MNDs. However, the intensity of these signals is small, which makes it possible to register the MNDs using EPR spectrometry with the necessary accuracy. The data obtained open up the prospects of using the EPR method for studies of the interorgan distribution, accumulation, and elimination of MNDs during their intravenous injection into experimental animals.

Fluorescent nanodiamonds engage innate immune effector cells: A potential vehicle for targeted anti-tumor immunotherapy.

Fluorescent nanodiamonds (FNDs) are nontoxic, infinitely photostable, and emit fluorescence in the near infrared region. Natural killer (NK) cells and monocytes are part of the innate immune system and are crucial to the control of carcinogenesis. FND-mediated stimulation of these cells may serve as a strategy to enhance anti-tumor activity. FNDs were fabricated with a diameter of 70±28 nm. Innate immune cell FND uptake, viability, surface marker expression, and cytokine production were evaluated in vitro. Evaluation of fluorescence emission from the FNDs was conducted in an animal model. In vitro results demonstrated that treatment of immune cells with FNDs resulted in significant dose-dependent FND uptake, no compromise in cell viability, and immune cell activation. FNDs were visualized in an animal model. Hence, FNDs may serve as novel agents with "track and trace" capabilities to stimulate innate immune cell anti-tumor responses, especially as FNDs are amenable to surface-conjugation with immunomodulatory molecules.

Nanodiamond-Manganese dual mode MRI contrast agents for enhanced liver tumor detection.

Contrast agent-enhanced magnetic resonance (MR) imaging is critical for the diagnosis and monitoring of a number of diseases, including cancer. Certain clinical applications, including the detection of liver tumors, rely on both T1 and T2-weighted images even though contrast agent-enhanced MR imaging is not always reliable. Thus, there is a need for improved dual mode contrast agents with enhanced sensitivity. We report the development of a nanodiamond-manganese dual mode contrast agent that enhanced both T1 and T2-weighted MR imaging. Conjugation of manganese to nanodiamonds resulted in improved longitudinal and transverse relaxivity efficacy over unmodified MnCl2 as well as clinical contrast agents. Following intravenous administration, nanodiamond-manganese complexes outperformed current clinical contrast agents in an orthotopic liver cancer mouse model while also reducing blood serum concentration of toxic free Mn2+ ions. Thus, nanodiamond-manganese complexes may serve as more effective dual mode MRI contrast agent, particularly in cancer.

Coherent anti-Stokes Raman scattering microscopy of single nanodiamonds.

Nanoparticles have attracted enormous attention for biomedical applications as optical labels, drug-delivery vehicles and contrast agents in vivo. In the quest for superior photostability and biocompatibility, nanodiamonds are considered one of the best choices due to their unique structural, chemical, mechanical and optical properties. So far, mainly fluorescent nanodiamonds have been utilized for cell imaging. However, their use is limited by the efficiency and costs in reliably producing fluorescent defect centres with stable optical properties. Here, we show that single non-fluorescing nanodiamonds exhibit strong coherent anti-Stokes Raman scattering (CARS) at the sp(3) vibrational resonance of diamond. Using correlative light and electron microscopy, the relationship between CARS signal strength and nanodiamond size is quantified. The calibrated CARS signal in turn enables the analysis of the number and size of nanodiamonds internalized in living cells in situ, which opens the exciting prospect of following complex cellular trafficking pathways quantitatively.

Range-finding risk assessment of inhalation exposure to nanodiamonds in a laboratory environment.

This study considers fundamental methods in occupational risk assessment of exposure to airborne engineered nanomaterials. We discuss characterization of particle emissions, exposure assessment, hazard assessment with in vitro studies, and risk range characterization using calculated inhaled doses and dose-response translated to humans from in vitro studies. Here, the methods were utilized to assess workers' risk range of inhalation exposure to nanodiamonds (NDs) during handling and sieving of ND powder. NDs were agglomerated to over 500 nm particles, and mean exposure levels of different work tasks varied from 0.24 to 4.96 µg·m(-3) (0.08 to 0.74 cm(-3)). In vitro-experiments suggested that ND exposure may cause a risk for activation of inflammatory cascade. However, risk range characterization based on in vitro dose-response was not performed because accurate assessment of delivered (settled) dose on the cells was not possible. Comparison of ND exposure with common pollutants revealed that ND exposure was below 5 µg·m(-3), which is one of the proposed exposure limits for diesel particulate matter, and the workers' calculated dose of NDs during the measurement day was 74 ng which corresponded to 0.02% of the modeled daily (24 h) dose of submicrometer urban air particles.

Multi-color imaging of fluorescent nanodiamonds in living HeLa cells using direct electron-beam excitation.

Multi-color, high spatial resolution imaging of fluorescent nanodiamonds (FNDs) in living HeLa cells has been performed with a direct electron-beam excitation-assisted fluorescence (D-EXA) microscope. In this technique, fluorescent materials are directly excited with a focused electron beam and the resulting cathodoluminescence (CL) is detected with nanoscale resolution. Green- and red-light-emitting FNDs were employed for two-color imaging, which were observed simultaneously in the cells with high spatial resolution. This technique could be applied generally for multi-color immunostaining to reveal various cell functions.

Aggregation behavior of nanodiamonds and their functionalized analogs in an aqueous environment.

The colloidal behavior of aqueous dispersions of detonation nanodiamonds (DNDs) and carboxylated nanodiamonds (DND-COOH) which were synthesized via a microwave process is presented. Both forms of DNDs were found to be relatively stable in aqueous solutions, but aggregated rapidly in the presence of mono and divalent salts. The critical coagulation concentration (CCC) values for DNDs and DND-COOH were estimated to be between 8 and 10 mM for NaCl and 7 and 8 mM for MgCl2. In general, the formation of carboxyl groups on the DND surface did not alter colloidal behavior as dramatically as it is known to do for other nanocarbons especially carbon nanotubes.

Biomedical applications of nanodiamonds in imaging and therapy.

Nanodiamonds have attracted remarkable scientific attention for bioimaging and therapeutic applications owing to their low toxicity with many cell lines, convenient surface properties and stable fluorescence without photobleaching. Newer techniques are being applied to enhance fluorescence. Interest is also growing in exploring the possibilities for modifying the nanodiamond surface and functionalities by attaching various biomolecules of interest for interaction with the targets. The potential of Raman spectroscopy and fluorescence properties of nanodiamonds has been explored for bioimaging and drug delivery tracing. The interest in nanodiamonds' biological/medical application appears to be continuing with enhanced focus. In this review an attempt is made to capture the scope, spirit and recent developments in the field of nanodiamonds for biomedical applications.

Evidence from central Mexico supporting the Younger Dryas extraterrestrial impact hypothesis.

We report the discovery in Lake Cuitzeo in central Mexico of a black, carbon-rich, lacustrine layer, containing nanodiamonds, microspherules, and other unusual materials that date to the early Younger Dryas and are interpreted to result from an extraterrestrial impact. These proxies were found in a 27-m-long core as part of an interdisciplinary effort to extract a paleoclimate record back through the previous interglacial. Our attention focused early on an anomalous, 10-cm-thick, carbon-rich layer at a depth of 2.8 m that dates to 12.9 ka and coincides with a suite of anomalous coeval environmental and biotic changes independently recognized in other regional lake sequences. Collectively, these changes have produced the most distinctive boundary layer in the late Quaternary record. This layer contains a diverse, abundant assemblage of impact-related markers, including nanodiamonds, carbon spherules, and magnetic spherules with rapid melting/quenching textures, all reaching synchronous peaks immediately beneath a layer containing the largest peak of charcoal in the core. Analyses by multiple methods demonstrate the presence of three allotropes of nanodiamond: n-diamond, i-carbon, and hexagonal nanodiamond (lonsdaleite), in order of estimated relative abundance. This nanodiamond-rich layer is consistent with the Younger Dryas boundary layer found at numerous sites across North America, Greenland, and Western Europe. We have examined multiple hypotheses to account for these observations and find the evidence cannot be explained by any known terrestrial mechanism. It is, however, consistent with the Younger Dryas boundary impact hypothesis postulating a major extraterrestrial impact involving multiple airburst(s) and and/or ground impact(s) at 12.9 ka.