The structural and surface properties of the nanodiamonds, sized 10 nm, were verified by high-resolution transmission microscopy (HRTEM), X-ray photoelectron spectroscopy (XPS), and Raman spectroscopy. The Raman images of the diamonds are shown in Figure S1A in Supplementary Material. The spectra reveal the characteristic feature of diamonds with a peak at 1336/cm. The presence of graphitic carbon with bands at 1350 and 1580/cm is virtually absent. The XPS survey spectra of the diamonds showed signals of carbon (10 nm: 90.4%; 1000 nm: 90.1%) as dominant element together with the presence of oxygen (10 nm: 4.2%; 1000 nm: 9.6%) and nitrogen (10 nm: 1.8%; 1000 nm: 0.3%). Figure S1B in Supplementary Material shows (high resolution) transmission microscopy pictures of 10 nm nanodiamonds with a diameter of about 7 ± 4 nm. The lattice fringes with a spacing of 2.06 Å are assigned to the diamond (111) plane. The physicochemical properties of the diamonds are listed in Table 1.

Freshly isolated human polymorphonuclear leukocytes (PMN) and peripheral blood mononuclear cells (PBMC) showed rapidly increasing membrane permeability, represented by an increase of the SYTOX Green signal due to its increased accessibility to DNA (Figure 1A) when exposed to 10 nm diamonds (nanodiamonds). SYTOX Green is a cell membrane-impermeable dye that specifically intercalates into accessible DNA, thereby increasing its fluorescence up to 500 times. Interestingly, the increase of the SYTOX signal in PBMC was less pronounced than the one observed in PMN. Next, we compared the SYTOX signal induced by nanodiamonds with the one induced by PMA as a classical stimulus for DNA externalization in conjunction with NETosis. In response to nanodiamonds, the SYTOX signal was enhanced earlier (60 min) and more pronounced than with PMA (Figure 1B). In contrast, upon incubation with the control 1000 nm diamonds (microdiamonds), the SYTOX signal was comparable to unstimulated cells, showing no membrane damage and no accessibility of the DNA in PBMC or PMN (Figure 1A). The membrane damage in response to nanodiamonds was dose dependent as represented by the increase of the SYTOX signal in PMN incubated with increasing amounts of particles (Figure 1C). We conclude that nanodiamonds induce rapid cell membrane rupture rendering DNA accessible in white blood cells.

In order to characterize the nuclear appearance of leukocytes upon contact with diamonds, we microscopically analyzed PMN and PBMC after co-incubation with nanodiamonds or microdiamonds. DNA was stained by propidium iodide or Hoechst 33342 and the DNA-associated proteins citH3 and NE by immunofluorescence. PMN incubated with nanodiamonds exhibited large spread aggregates composed of DNA and nanodiamonds co-localizing with citH3 and NE (Figure 1D). In contrast, employing microdiamonds no such structures were observed. The nuclei displayed a lobular shape, characteristic of neutrophils, and the signals of NE and citH3 were localized intracellularly. The nuclear appearance of PBMC incubated with nanodiamonds differed strongly from that of similarly treated PMN (Figure 1E). Only sporadic diamond and necrotic cell aggregates with normal nuclear morphology were observed in association with nanodiamonds. NET-like structures trapping nanodiamonds were not observed in PBMC samples. Incubation with microdiamonds induced nuclear modifications neither in PBMC nor in PMN (Figures 1D,E).

Live cell imaging confirmed the fast and uncontrolled rupture of the plasma membrane of PMN (Video S1 in Supplementary Material) and PBMC (Video S2 in Supplementary Material) in response to nanodiamonds. This process is represented by the conversion of the blue Hoechst3342 signal, being cell membrane permeable, to the red PI signal, intercalating into accessible DNA. The nuclear appearance of PMN markedly differed from that of PBMC. The lobulated nucleus of viable neutrophils, stained by Hoechst3342, became PI-positive, and displayed a decondensed morphology as soon as 30 min after the stimulus (Video S1 in Supplementary Material). This process was followed by externalization and spreading of the DNA. In contrast, nuclei of PBMC quickly became PI-positive, indicative for plasma membrane damage, but the DNA was not externalized and spread (Video S2 in Supplementary Material). Microdiamonds associated with both, PMN (Video S3 in Supplementary Material) and PBMC (Video S4 in Supplementary Material), but did not affect the integrity of their cellular membranes. However, some spontaneous cell death was observed most likely due to phototoxicity in time-lapse fluorescence microscopy (27).

Quantification of the signal for extracellular DNA and citH3 revealed a significant increase when PMN were incubated with nanodiamonds in comparison to microdiamonds or unstimulated cells (Figure 1F). Externalized chromatin of neutrophils co-localizing with citH3 is indicative for NETosis (11). In order to further evaluate NETosis, we added nanodiamonds to PMN pre-incubated with the ROS scavenger N-acetyl l-cysteine (NAC). Although we still observed NET formation in the presence of NAC, the aggregation of the nanodiamonds by NETs was reduced when compared to that resulting from the incubation of PMN with nanodiamonds alone (Figure 1G). This was confirmed by in silico morphometric quantification of NET aggregation concerning the number and area of NET structures (Figure 1H). In the presence of the ROS scavenger, the number of NET aggregates (AggNET) was significantly higher, but the area was significantly decreased. This reflects a reduced aggregation of NETs in the absence of ROS. Next, we employed CFSE-labeled PBMC, which had previously been incubated with nanodiamonds to stimulate freshly isolated viable PMN. Interestingly, we observed that necrotic PBMCs and nanodiamonds were entrapped in the NET aggregates characterized by large DNA filaments decorated with NE and citH3 (Figure 1I).

We conclude that nanodiamonds induced fast rupture of the plasma membrane when encountering leukocytes (PMN and PBMC). The mononuclear cells rapidly died by membrane rupture and became necrotic. Contrarily, PMN formed NETs, which tended to aggregate in the presence of ROS to confine nanodiamonds as well as necrotic mononuclear cells.

In order to determine the effect of nano- and microdiamonds on murine leukocytes, we quantified the accessibility of DNA for SYTOX Green in isolated bone marrow cells. To further analyze the role of the oxidative burst on nanoparticle-induced NETosis, experiments were conducted with bone marrow cells of both, WT and Ncf1** mice. Latter harbor a single-nucleotide polymorphism in the gene for the regulatory p47^phox subunit (Ncf1) of the NADPH oxidase NOX2. This mutation leads to strongly diminished NOX2-dependent ROS production resulting in a deficiency of neutrophils to undergo NOX2-dependent NETosis.

Isolated bone marrow cells contained about 80% CD45⁺ leukocytes. More than 50% of these were identified as granulocytes (Figures S1C,D in Supplementary Material). Consistent with previous experiments employing human leukocytes, we observed a fast permeabilization of the plasma membrane in response to nanodiamonds in bone marrow cells of both, WT and Ncf1** mice (Figure 2A), while the microdiamonds were inert. Stimulation with ionomycin of bone marrow cells triggered DNA release in both, WT and Ncf1** mice (Figure 2B). This stimulus reportedly induces NETosis independent of NOX2-dependent ROS production (28). Importantly, only leukocytes from WT mice underwent DNA externalization in response to PMA, a NOX2-dependent ROS inducing stimulus (29), whereas Ncf1**-derived cells did not. Similar to human cells, the quantification of extracellular citH3 revealed significantly elevated signals after exposure to nanodiamonds in comparison to microdiamonds or unstimulated cells (Figures 2C,D). Analyses by microscopy of WT or Ncf1** bone marrow cells incubated with nanodiamonds revealed a similar appearance to that of human PMN (Figures 2E,F). The nanodiamonds induced NET structures of DNA co-localizing with citH3 or NE and sequestering the particles in both, WT- and Ncf1**-derived cells (Figures 2E,F). The microdiamonds did not induce the formation of such structures (Figures 2E,F). Lower magnification pictures showing NET structures or nuclear morphology in a larger area are depicted in Figures S1E,F in Supplementary Material. From these observations, we can conclude that the direct effects of nanodiamonds on both human and murine leukocytes are ROS independent.

Nanodiamonds induced rapid and substantial cellular damage in vitro independently of the leukocyte type in both human and mice. Since nanodiamonds cannot be digested enzymatically, we hypothesized that their persistent presence might result in a continuous induction of inflammation in vivo. To investigate the role of NOX2-dependent NETosis in nanoparticle-induced inflammation, we injected 1 mg of nanodiamonds or microdiamonds into the metatarsal region of the hind paws of WT or Ncf1** mice. Paw edema in the particle-injected foot was recorded as a specific sign of local inflammation over 28 days and compared to the sham-treated control foot. Already 24 h after injection, we observed the development of significant paw edema in both mouse strains injected with nanodiamonds (Figure 3A). In WT mice, the inflammation resolved within 3 days. Ncf1** mice developed a sustained inflammation that did not resolve until the end of the experiment at day 28. In contrast, microdiamonds neither induced paw swelling in WT nor in Ncf1** mice (Figure 3B). In an additional experiment, we injected nanodiamonds in the presence of DNase I into WT mice (Figure 3C). To avoid further injection-induced tissue damage, DNase I was applied only once together with the nanodiamonds as well as i.v. 24 and 48 h after injection of nanodiamonds. Paw edema was recorded for 28 days. Since no differences between the groups at later time points were observed, values until day 10 are shown. DNase I-treated mice showed prolonged inflammation until day 5, while in the untreated group, the inflammation resolved already at day 4.

Dissection of the WT hind paws disclosed that nanodiamonds were wrapped in membrane-like structures resembling granulation tissue (Figure 3D). In contrast, Ncf1** hind paws showed bare nanodiamonds without visible association to connective tissue. The microdiamonds injected into hind paws showed no signs of clumping or granulation (Figure 3D). Moreover, nanodiamonds were tightly attached to the overlaying skin in WT paws, while they appeared more loose and spread in the surrounding tissues in the paws of Ncf1** mice (Figure 3E). Histological analysis of the skins revealed packing of nanodiamonds associated with DNA and NE in WT mice (Figure 3F). In contrast, nanodiamonds were dispersed in the skin section and not associated with extracellular neutrophil markers in Ncf1** mice.

In summary, nanodiamonds trigger a strong local inflammatory response when injected into tissues by inducing membrane rupture and necrosis. In WT mice, cell death in response to nanodiamonds induced ROS-dependent NETosis, organization of nanodiamonds in the tissue, and resolution of the initial inflammation. In the absence of NOX2-dependent NETosis; however, inflammation does not resolve, nanodiamonds are dispersed in the tissue, and this may trigger chronic inflammation.

All analyses of human material were performed in full agreement with institutional guidelines and with the approval of the Ethical committee of the University Hospital Erlangen (permit # 193 13B). Human peripheral PMN and PBMC were isolated from heparinized (20 U/ml) venous blood of normal healthy donors (NHD) by Lymphoflot (Bio-Rad, Hercules, CA, USA) density gradient centrifugation as described elsewhere (50). Briefly, whole blood was carefully pipetted on Lymphoflot solution and centrifuged for 30 min at 1400 rpm. Then, the plasma was carefully removed and the PBMC layer was collected. The PMN-rich layer on top of RBC was taken and subjected to hypotonic lysis of RBC. Cell viability was assessed by trypan blue exclusion.

Ncf1** mice, harboring a single-nucleotide polymorphism in the gene for the regulatory p47phox subunit (Ncf1) of the NADPH oxidase NOX2 (51, 52), originate from The Jackson Laboratories and were backcrossed over more than 10 generations to the BALB/c background and maintained at the animal facilities of the University of Erlangen. The animal studies were approved by the Veterinary Office of the Government of lower Franconia (permit # 55.2 DMS-2532-2-103) and conducted according to the guidelines of the Federation of European Laboratory Animal Science Associations (FELASA). Genotyping of Ncf1** and WT littermates was done by pyrosequencing, as described (53).

Transmission electron microscopy (TEM) images were recorded on a JEOL JEM-2011 electron microscope operated at an accelerating voltage of 200 kV. XPS measurements were performed with an ESCALAB 220 XL spectrometer from Vacuum Generators featuring a monochromatic Al Kα X-ray source (1486.6 eV) and a spherical energy analyzer operated in the CAE (constant analyzer energy) mode (CAE = 100 eV for survey spectra and CAE = 40 eV for high-resolution spectra), using the electromagnetic lens mode. No flood gun source was needed due to the conducting character of the substrates. The angle between the incident X-rays and the analyzer is 58°. The detection angle of the photoelectrons is 30°. Zeta (ζ) potential measurements were performed with a Zetasizer Nano ZS (Malvern Instruments S.A., Worcestershire, UK). The pH of all the samples was maintained at ~7.4. Micro-Raman spectroscopy measurements were performed on a Horiba Jobin Yvon LabRam HR micro-Raman system combined with a 473 nm (1 mW) laser diode as excitation source. Visible light is focused by a 100× objective. The scattered light is collected by the same objective in backscattering configuration, dispersed by a 1800-mm focal length monochromator and detected by a CCD camera. In order to exclude endotoxin contamination, diamonds were treated with NaOH, dried out in ethanol, and treated with 300°C heat before used in cultures and animal experiments.

We injected 1 mg of 10 or 1000 nm diamonds in 70 μl 0.9% sterile NaCl solution into the metatarsal region of the hind paws of Ncf1** and WT mice. The contralateral paw was injected with 70 μl 0.9% NaCl solution serving as control treatment. In order to assess the role of NETs in the resolution of nanoparticle-induced inflammation, we injected 1 mg of 10 nm diamonds in 70 μl 0.9% sterile NaCl solution containing 200 μg DNase I (Sigma-Aldrich) into the metatarsal region of the hind paws of WT mice. The contralateral paw was injected with 70 μl 0.9% NaCl solution containing 200 μg DNase I serving as control treatment. Additionally, 500 μg DNase I was injected intravenously 24 and 48 h after particle injection. To monitor paw edema as a sign of inflammation, foot pads were measured with an electronic caliper at the indicated time points. On day 28, mice were sacrificed by CO₂ aspiration. Some mice were sacrificed at day 15 and hind paws were fixed in 4% paraformaldehyde then transferred to 70% ethanol for dissection.

Bone marrows of femurs and tibiae of BALB/c WT and Ncf1** mice were flushed out with PBS and RBC were lysed using Tris-buffered (0.15 M) ammonium chloride (0.16 M) adjusted to pH 1.65.

Freshly isolated human PMN and PBMCs or murine bone marrow cells were adjusted to a concentration of 2 × 10⁶ cells/ml in HBSS containing 5.55 mM glucose, 1.2 mM calcium, and 0.5 mM magnesium (Thermo Fisher Scientific) and plated in 96-well plates (Greiner Bio-One, Frickenhausen, Germany) in a final cell density of 1 × 10⁶ cells/ml. The DNA dye SYTOX Green (Thermo Fisher Scientific) was added in a final concentration of 2.5 μM. Diamonds of 10 and 1000 nm were added in a final concentration of 200 μg/ml or as indicated. The traditional NETosis-inducing stimuli ionomycin (InvivoGen, San Diego, CA, USA) or PMA (Sigma, Darmstadt, Germany) were added in final concentrations of 1 μg/ml or 100 ng/ml, respectively. Plates containing PMA, ionomycin, 10 or 1000 nm diamonds in 100 μl of HBSS, respectively, were incubated at 37°C and 5% CO₂ prior to addition of cells to equilibrate pH. Living cells were added and plates were covered. DNA externalization was analyzed for 210 min. on an Infinite^® 200 PRO plate reader (TECAN, Crailshaim, Germany) under controlled temperature. Excitation was performed at 485 nm and emission was detected at 535 nm. Values displayed in the graphs were normalized according to absorption or addition of fluorescence by 10 or 1000 nm diamonds, respectively.

For microscopic analysis, 200,000 per well freshly isolated PMN or PBMC or 600,000 murine bone marrow cells per well were seeded in RPMI (Thermo Fisher Scientific, Waltham, MA, USA) in LabTek2 Chamber Slides (Thermo Fisher Scientific, Waltham, MA, USA). After addition of 200 μg/ml, 10 or 1000 nm diamonds (Sigma-Aldrich, St. Louis, MO, USA), 10 μg/ml PMA (Sigma-Aldrich, St. Louis, MO, USA), or 1 μg/ml ionomycin (Sigma-Aldrich, St. Louis, MO, USA) cells were incubated for 4 h at 37°C and 5% CO₂. For experiments using the ROS scavenger N-acetyl l-cysteine (NAC) (Sigma-Aldrich, St. Louis, MO, USA), PMN were pre-incubated for 30 min at 37°C and 5% CO₂ with 5 mM NAC for 30 min before addition of 200 μg/ml 10 nm nanodiamonds and incubation for 4 h at 37°C and 5% CO₂. For experiments with CFSE-labeled cells, freshly isolated PBMC were labeled with CellTrace™ CSFE (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Then, cells were fixed with 0.1% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 15 min and stained, as described below.

For analysis of murine tissue samples, histology was performed using paraffin embedding. Tissue samples were fixed overnight in 4% formalin, dehydrated with ethanol, and subsequently embedded in paraffin. This was followed by immunofluorescence staining. Chamber slides and deparaffinized sections were blocked for 18 h at 4°C with PBS containing 10% FBS (Merck Millipore, Billerica, Waltham, MA, USA). Primary antibodies detecting NE (ab21595, Abcam, Cambridge, UK) or citH3 (ab5103, Abcam, Cambridge, UK) were added to the slides in a 1:200 dilution and incubated for 1.5 h at room temperature. This was followed by incubation with the Cy5-conjugated secondary detection antibody AffiniPure Goat Anti-Rabbit IgG (H + L) (Jackson Immuno Research Labs, West Grove, PA, USA) in a dilution 1:400 for 1 h at room temperature in the dark together with Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA). Slides were washed with PBS and H₂O and samples were embedded in DAKO fluorescent mounting medium (Agilent Technologies, Santa Clara, CA, USA). Slides were analyzed either using the Eclipse Ni-U (Nikon Corporation, Tokyo, Japan) or the BZ-X700 microscope (Keyence Corporation, Osaka, Japan). Z-stacks were performed to increase depth of field. Post-processing of pictures was performed using Photoshop CS5 (Adobe, München, Germany).

The 200,000 PMN or PBMC or murine bone marrow cells resuspended in RPMI were seeded in poly l-lysine (Sigma-Aldrich, St. Louis, MO, USA)-coated 96-well flat bottom cell culture plates. Cells were then incubated with 200 μg/ml 10 or 1000 nm diamonds, 10 μg/ml PMA (Sigma-Aldrich, St. Louis, MO, USA), or 1 μg/ml ionomycin (Sigma-Aldrich, St. Louis, MO, USA) for 4 h at 37°C, and 5% CO₂. After incubation, DNA was stained with the membrane-impermeable DNA dye DRAQ7 (BioLegend, San Diego, CA, USA) at room temperature for 15 min. After centrifugation for 5 min at 1800 rpm, samples were measured using the near-infrared fluorescence imaging system Odyssey^® CLx Imaging System (LI-COR, Lincoln, NE, USA) at 700 nm. After DNA measurement, cells were fixed with 0.1% paraformaldehyde for 15 min and blocked for 18 h at 4°C in PBS containing 10% FBS. Samples were incubated with the primary antibody recognizing citH3 for 1.5 h at room temperature, followed by incubation with the secondary goat anti-rabbit detection antibody IRDye^® 800 CW (LI-COR, Lincoln, NE, USA) for 1 h at room temperature. After centrifugation for 5 min at 1800 rpm, samples were measured with the Odyssey^® Clx Imaging System at 800 nm. Post-processing of pictures and quantification of the signals was performed using Photoshop CS5 (Adobe, München, Germany).

Isolated PMN and PBMC were adjusted to a concentration of 1 × 10⁶ cells/ml in RPMI. Cell suspensions were added to an 8-well Nunc chamber slide (VWR, Darmstadt, Germany). Chamber slides were pre-incubated at 37°C at least 30 min prior to addition of 200 μl of 10 or 1000 nm diamonds with a concentration of 200 μg/ml each. Staining solution containing 0.1 μg/ml Hoechst 33342 and 1 μg/ml PI in PBS was added shortly before addition of the diamonds. Slides were analyzed on a BZ-X710 microscope (Keyence, Neu-Isenburg, Germany). Z-stacks were performed to increase depth of field. Post-processing of pictures was performed with Photoshop CS5 (Adobe, München, Germany).

Results are represented as the mean ± SEM of the indicated number of replicates/experiments. We performed computations and created charts using the GraphPad Prism 5.03 software. For calculation of statistical differences among the groups, we used ANOVA test with Bonferroni post hoc correction, where applicable. Adjusted p-values <0.05 were considered to be statistically significant.