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Fluorescent Nanodiamonds for Tracking Single Polymer Particles in Cells and Tissues
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Fluorescent Nanodiamonds for Tracking Single Polymer Particles in Cells and Tissues
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  • Runrun Li
    Runrun Li
    Department of Biomedical Engineering, Groningen University, University Medical Center Groningen, Antonius Deusinglaan 1, 9713AV Groningen, The Netherlands
    More by Runrun Li
  • Thea A. Vedelaar
    Thea A. Vedelaar
    Department of Biomedical Engineering, Groningen University, University Medical Center Groningen, Antonius Deusinglaan 1, 9713AV Groningen, The Netherlands
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  • Alina Sigaeva
    Alina Sigaeva
    Department of Biomedical Engineering, Groningen University, University Medical Center Groningen, Antonius Deusinglaan 1, 9713AV Groningen, The Netherlands
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  • Yue Zhang
    Yue Zhang
    Department of Biomedical Engineering, Groningen University, University Medical Center Groningen, Antonius Deusinglaan 1, 9713AV Groningen, The Netherlands
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  • Kaiqi Wu
    Kaiqi Wu
    Department of Biomedical Engineering, Groningen University, University Medical Center Groningen, Antonius Deusinglaan 1, 9713AV Groningen, The Netherlands
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  • Hui Wang
    Hui Wang
    Zernike Institute for Advanced Materials, Groningen University, Nijenborgh 4, 9747 AG Groningen, The Netherlands
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  • Xixi Wu
    Xixi Wu
    Department of Biomedical Engineering, Groningen University, University Medical Center Groningen, Antonius Deusinglaan 1, 9713AV Groningen, The Netherlands
    Zernike Institute for Advanced Materials, Groningen University, Nijenborgh 4, 9747 AG Groningen, The Netherlands
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  • Peter Olinga
    Peter Olinga
    Department of Pharmaceutical Technology and Biopharmacy, Groningen University, University Medical Center Groningen, Antonius Deusinglaan 1, 9713AV Groningen, The Netherlands
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  • Małgorzata K. Wlodarzyk-Biegun
    Małgorzata K. Wlodarzyk-Biegun
    Zernike Institute for Advanced Materials, Groningen University, Nijenborgh 4, 9747 AG Groningen, The Netherlands
    Biotechnology Centre, The Silesian University of Technology, Krzywoustego 8, 44-100 Gliwice, Poland
    More by Małgorzata K. Wlodarzyk-Biegun
  • Romana Schirhagl*
    Romana Schirhagl
    Department of Biomedical Engineering, Groningen University, University Medical Center Groningen, Antonius Deusinglaan 1, 9713AV Groningen, The Netherlands
    *Email: [email protected]
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    Orcidhttps://orcid.org/0000-0002-8749-1054
Open PDFSupporting Information (1)

Analytical Chemistry

Cite this: Anal. Chem. 2023, 95, 35, 13046–13054
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https://doi.org/10.1021/acs.analchem.3c01452
Published August 23, 2023

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Abstract

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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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Introduction

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Polymer nanoparticles are widely used for drug delivery. (1−3) It is important to understand their interactions with biological samples to assess nanoparticle toxicity. (4) Further, it is essential to know how polymer particles, produced by degrading plastic waste in the environment, interact with biological matter. (5) For all of these applications, it is vital to study where within cells or tissues polymer nanoparticles exactly end up. There are several imaging approaches to visualize polymer particles. (6) Electron microscopy is an attractive tool to achieve this goal. (7) However, electron microscopy is usually not compatible by analyzing living samples. Additionally, most polymers are relatively similar in elemental composition to biological matter, and thus, this is especially challenging for small particles. Optical microscopy and spectroscopy offer an alternative, but most polymer particles cannot be detected directly with these approaches either. (8,9) To circumvent this problem, it is possible to include fluorescent labels inside the polymer. However, conventional fluorescent labels suffer from bleaching and can thus not be followed in the long term.
Fluorescent nanodiamonds (FNDs) offer an attractive alternative to conventional fluorescent labels. FNDs are excellently biocompatible in different kinds of cells and even in entire organisms. (10−13) Further, FNDs are inert but can be chemically functionalized via their rich surface chemistry. Most importantly, FNDs can host color centers that are protected within the diamond crystal lattice and emit infinitely stable fluorescence. Making use of these properties, FNDs have been utilized for long-term fluorescent labeling as well as tracking. (14−17) By attaching antibodies, glycans, or targeting peptides, it is possible to target nanodiamonds to specific regions within the cell or tissue to visualize these structures. (18−20) It is also attractive that FNDs are well visible in different imaging techniques, which are interesting for correlative microscopy. (21) Finally, nanodiamonds can be used for quantum sensing to determine the particle orientation, (22) temperature, (23,24) or the free radical load in their surroundings. (25−28) Here, we use these FNDs for the first time to track where polymer particles reside within cells or tissues.
The study scheme, including particle preparation and in vitro measurements, is shown in Figure 1.

Figure 1

Figure 1. Preparation of PLA-FND particles, in vitro evaluation, and cellular tracking. (a) PLA and FNDs in chloroform are added to PVA in aqueous solution. (b) Tip sonication facilitates the formation of an emulsion, and the presence of PVA stabilizes the emulsion and leads to a uniform particle size distribution. (c) Particles were collected by centrifugation after magnetic stirring overnight. (d) Particle internalization and location within the cell and quantitative analysis. (e) Particle internalization in mouse liver slice. (f) Diamonds within polymer particles were tracked using a custom-built confocal microscope.

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Materials and Methods

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Throughout this study, we use FNDs of an average hydrodynamic diameter of 70 nm (commercially available from Adamas Nanotechnologies). These FNDs are produced by high-pressure high-temperature synthesis by the manufacturer as described before. (29) After that, they are irradiated with high-energy electrons at 3 MeV and a fluence of 5 × 1019 e/cm2, followed by a high-temperature annealing step. Finally, FNDs are cleaned by the manufacturer in oxidizing the acid to obtain oxygen-terminated FNDs. The diamond particles themselves are widely used and well characterized in the field. (30,29)
Polylactic acid (PLA, Mw 60,000), chloroform (≥99.5%), poly(vinyl alcohol) (PVA,87–90% hydrolyzed, average mol wt 30,000–70,000), 4,6- diamidino-2-phenylindole (DAPI), phalloidin–fluorescein isothiocyanate labeled (phalloidin-FITC), and lysosome-associated membrane protein 1(LAMP-1, AB2971) were all purchased from Sigma-Aldrich, The Netherlands. 4% formaldehyde solution was obtained from Klinipath, Poland, and early endosomal antigen 1 (EEA1, # MA5-14794) from Thermofisher, The Netherlands. Donkey-α-rabbit IgG FITC was purchased from Jackson, U.K. (711-095-152). Dulbecco’s Modified Eagle Medium with high glucose (DMEM-HG, 31966047), fetal bovine serum (FBS, 10270106), and penicillin–streptomycin (10,000 U/mL, 15140122) were obtained from Gibco, The Netherlands. The adherent mouse macrophage cell line, J774, was obtained from ATCC/LCG and cultured in DMEM-HG supplemented with 10% (v/v) FBS, 100 units/mL penicillin, and 100 mg/mL streptomycin in a humidified 5% CO2/95% air atmosphere at 37 °C.

Synthesis and Characterization of nanoparticles

PLA nanoparticles and PLA NPs loaded with FNDs (PLA-FNDs) were prepared by a classic solvent emulsion evaporation method. (31) Appropriate amounts of PLA were dissolved in chloroform to 10 mg/mL, and PVA was dissolved in Milli-Q water at 60 °C to 1% w/v, which is used here as the dispersant to control particle size and reduce aggregation. Then, the PLA solution was added to an aqueous solution of PVA (1:6 v/v PLA solution and PVA solution). For PLA nanoparticles loaded with FNDs, 70 nm FNDs (2%, w(FNDs)/w(PLA)) were mixed with the PLA solution in chloroform first and then added into the aqueous solution of PVA. The mixture was tip-sonicated (Vibra-Cells, VCX130, 2 mm stepped micro tip) for 3 min in a pulsed manner and 40% amplitude over an ice bath. The emulsion was magnetically stirred overnight, while chloroform was evaporated at room temperature. Subsequently, nanoparticles were collected by centrifugation at 10,000 rpm for 15 min in a pellet. We removed the solvent and resuspended the particles in a Milli-Q water. To wash the particles and remove residual PVA, we centrifuged again and removed the solvent. Washing was repeated 3 times in total. Nanoparticles were resuspended in water and stored at 4 °C.
The morphology of the nanoparticles was observed with a scanning electron microscope (SEM, FEI Nova NanoSEM 650). We dropped 10 μL of a 2 mg/mL nanoparticle suspension onto small silicon wafers and left the samples to dry. Images were taken under high vacuum at an acceleration of 10 kV. Particle size distributions and zeta potentials were determined by a Zetasizer Nano ZS ZEN3600 system (Malvern Instruments, U.K.) at 25 °C (see the Supporting Information). The biocompatibility of the particles was confirmed using an XTT assay (Supporting Information).

Cellular Uptake of FNDs and PLA-FND Nanoparticles

J774 cells were seeded in a 35 mm Petri dish with four compartments after reaching 70–90% confluency at a density of 5000 cells/compartment and left for 24 h. Cells were incubated with 4 μg/mL FNDs or 200 μg/mL PLA-FNDs (the same amount of FNDs as in PLA-FNDs theoretically) for 2 h. Then, we replaced the medium with fresh medium without particles and incubated for 0, 6, and 24 h. The cells incubated with PLA nanoparticles and cells without particles served as controls. Cells were fixed with 4% paraformaldehyde for 15 min. Fixed cells were permeabilized with 0.5% Triton X-100 in PBS for 3 min. Then, the cytoskeleton was stained with 2 μg/mL of phalloidin-FITC for 30 min in the dark. Nuclei were stained with 2 μg/mL DAPI for 10 min. Cells were stored at 1% paraformaldehyde for imaging. Nanoparticles were visualized by using a Zeiss LSM780 confocal microscope. Filter settings for DAPI, phalloidin-FITC, and FNDs were 408/461, 488/525, and 561/650 nm, respectively.

Tissue Slicing and Uptake of FNDs and PLA-FND Nanoparticles

Animal experiments and the use of tissue were approved by the Animal Ethical Committee of the University of Groningen (CCD AVD10500202216104) and were performed in accordance with EU Directive 2010/63/EU for animal experiments. The protocol of precision-cut liver slices was already described in previous work. (32) Briefly, excised mouse liver and cylindrical cores were stored at the University of Wisconsin (UW) organ preservation solution (DuPont Critical Care, Illinois). The cores were prepared by using a 5 mm biopsy punch. Approximately 250 μm thick slices were prepared using a Krumdieck Live Tissue Microtome-MD6000 (Alabama Research and Development, Munford, AL). Ice-cold Krebs–Henseleit buffer 1× supplemented with 25 mM d-glucose (Merck, Darmstadt, Germany), 25 mM NaHCO3 (Merck), and 10 mM 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (MP Biomedicals GmbH, Germany) saturated with 95% O2 and 5% CO2 were used to collect slices. Then, liver slices were cultured in William’s E medium with GlutaMAX (Life Technologies, Carlsbad) supplemented with 2.75 μg/mL d-glucose monohydrate (Merck, Darmstadt, Germany) and 50 μg/mL gentamicin (Invitrogen, Paisley, U.K.) at 37 °C and 5% CO2 and 80% O2. 50 μg of FNDs and 2.5 mg of PLA-FNDs (the same amount of FNDs in PLA-FNDs as for bare FNDs) were added separately to 500 μL William’s E medium in each well of a 12-well plate. Liver slices were incubated with the medium containing a suspension of particles up to 48 h, and this medium was refreshed every 24 h. Liver slices were fixed for 15 min with 2% formaldehyde and imaged using a Zeiss LSM780 confocal microscope connected with a two-photon laser at an 850 nm excitation wavelength.

Cellular Distribution of FNDs and PLA-FND Nanoparticles

To verify the intracellular distribution of FNDs and PLA-FND nanoparticles, endosome and lysosome staining were performed. The same protocol as that described above was used for cellular uptake. After incubation with FNDs and PLA-FNDs for 0, 6, and 24 h, the cells were fixed and washed with cold PBS three times. Then, cells were permeabilized with 0.5% Triton X-100 for 15 min and washed with PBS three times for 5 min each. 1% BSA (bovine serum albumin) in PBS (PBSA) was added to cells as a blocking agent to bind nonspecific sites that interfere with antigen binding. Afterward, cells were stained with EEA1 in PBSA (early endosome marker, dilution 1:200) and LAMP-1 in PBSA (late endosomes and lysosomes marker, dilution 1:500) for 1 h at room temperature in the dark. Cells were washed with PBS three times for 5 min each and incubated with a secondary antibody (Donkey-α-rabbit IgG FITC in PBSA) for both primary antibodies (dilution 1:200). The cells were incubated for 1 h at room temperature in the dark. Then, cells were washed with PBS three times for 5 min each again and stored at 1% paraformaldehyde for imaging (Zeiss LSM700 confocal microscope). Filter settings of FITC and FNDs were 488 nm/525 nm and 561/650 nm, respectively.

Imaging Acquisition and Processing

3D z-stack images were obtained with a 63 × 1.3 oil objective, and all fluorescence signals were collected with 200 × 200 × 200 nm3 per pixel to record particle internalization and their spatial distribution. All images of cells were deconvoluted by two plugins of FIJI software: the “PSF generator” and “DeconvolutionLab2”. The “PSF generator” plugin is used to generate a realistic point spread function (PSF) and correct blurred representations of the actual object due to diffraction. The “DeconvolutionLab2” plugin is a mathematical inverse method that represents the original signal of imaging by making use of the PSF. The ″3D object counter” plugin is used to calculate the number of intracellular particles in z-stack images. The deconvolved images of the EEA1 and LAMP-1 channels were transformed into 3D Euclidean distance maps of the proteins. If the minimum distance of particles from the 3D Euclidean distance map was 0, then the particles were considered to be colocalized with the endosomes or lysosomes. All image analyses were performed using FIJI software.

Statistics

For all experimental data, we performed statistical evaluation by GraphPad Prism 8.0. To determine statistical significance, we used the Kruskal–Wallis test of one-way ANOVA to compare three or more groups. Data were presented as the mean ± standard deviation. The statistical results are shown as *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, and ns indicates nonsignificance. Confocal images were observed and analyzed by ZEN 3.3 and FIJI software.

Results and Discussion

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Cellular and Tissue Uptake

To study the interaction between PLA and the cells, we incubated macrophages with FND-PLA and FND for comparison. Figure 2 shows the results of these experiments. Already from these images, it is clear that macrophages ingest both FNDs and PLA-FNDs. When we compare these images with the control with PLA only, we do not see any red particles. This indicates that the particles that we see are indeed FNDs. This can be further confirmed by the fact that FNDs do not bleach and show perfectly stable fluorescence.

Figure 2

Figure 2. FNDs, PLA-FNDs, and PLA nanoparticles were taken up by macrophages after 2 h of incubation with particles plus 0, 6, and 24 h of incubation in the medium without particles. Red represent the FNDs, green represents the cytoskeleton, and blue represents the nucleus. Merged images are shown. The red fluorescence of FNDs and PLA-FNDs was seen after nanoparticles were taken up. In the PLA samples, we did not observe any red fluorescence inside the cells, indicating that PLA itself has no autofluorescence across the visible wavelength we used.

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To quantify the particle numbers that we see per cell, we performed particle counting. The results are shown in Figure 3A. From this experiment, we can draw several conclusions. The number of FNDs or FND-PLA molecules per cell decreases with time. Further, we can see that FNDs are ingested to a larger extent than FND-PLA. Many studies have demonstrated that particle size is one of the key factors for cellular uptake. Some studies indicated that higher uptake was observed for smaller nanoparticles and medium uptake for medium-sized particles. (33) However, some studies also indicated that smaller or larger particles could decrease the uptake rate; there is an optimum size, around 50 or 100 nm, where the uptake rate is the highest. In Figure 3A, there was no significant difference in the number of particles per cell either PLA-FNDs or FNDs between 0 and 6 h, but there was a significant difference between the early time points and 24 h. The number of cells barely increased during incubation from 0 to 6 h, but there was a significant increase after incubation of 24 h, with approximately twice as many cells in the observed region as after 0 h and 6 h, as shown in Figure 3C. Studies have shown that particles are divided among daughter cells during the cell division, which is likely responsible for the number of particles per cell decreasing after 24 h incubation. (34,35) Figure 3B demonstrates the aggregation of the particles inside the cell by analyzing the particle volume. We found that FNDs aggregate (or otherwise end up in the same location) more inside the cell than PLA-FNDs, as the volume of FND aggregation is approximately twice that of PLA-FNDs. This is probably due to the transport and storage of particles by the cells in the same locations. Here, it has to be noted that the volumes that are shown are only from the bright part of the particle (the polymer itself is invisible) since the PLA part of PLA-FNDs is invisible. Aggregation of either PLA-FNDs or FNDs was most notable at 6 h, and the volume of PLA-FNDs did not change significantly after 24 h of incubation, but the volume of FNDs became significantly smaller. FNDs have been shown to undergo aggregation when they are exposed to the medium or end up together when they are ingested in endosomes and/or lysosomes, but aggregation does not affect the NV center fluorescence of each diamond. (36) Polymer particles might also aggregate due to the surface energy or particle size. Here, only polymer nanoparticles loaded with nanodiamonds were considered because nanodiamonds have unique optical stability for long-term tracking.

Figure 3

Figure 3. Number of particles in each cell decreases over time; however, noticeable aggregation occurred. All data were analyzed from at least 50 cells. (A) The spatial distribution of particles per cell is shown in violin plots. Wider sections mean a higher probability that the number of particles per cell is distributed there. Black dotted lines from top to bottom mean third quartile, median, and first quartile, respectively. (B) The particle volume is shown in box plots. The box covers the 5–95 percentile of the data, and the horizontal lines that split the box are the median. (C) Cell numbers at varying incubation times are shown in scatter plots. Black lines are mean ± standard deviation.

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Figure 4

Figure 4. PLA-FNDs (A) and FNDs (B) were also observed in mouse liver tissue after 48 h of incubation (red: FNDs, green: tissue autofluorescence).

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To demonstrate labeling in tissue samples, we used 250 μm thick precision-cut liver slices (PCLS). These are an attractive alternative to experiments on living animals, which are limited for ethical reasons. If slices are used, then one can test multiple conditions in slices from the same animal. This reduces the differences in biological variability and reduces the number of required animals. Images were taken by two-photon microscopy, which has a longer wavelength and a lower energy but deeper tissue penetration than single-photon measurements. Figure 4 shows that both FNDs and PLA-FNDs can be taken up by the PCLS. Also, in tissue, FNDs seem to be easier to detect and more numerous. Red fluorescence of FNDs from PLA-FNDs was observed both at the edges and inside the tissue. Green fluorescence is tissue autofluorescence because of endogenous tissue components, such as the aromatic amino acids, collagens, nicotinamide adenine dinucleotide, retinol, and riboflavin. The concentrations of those intrinsic fluorophores are extremely high in tissue depending on tissue type. (37,38) The results reveal that 2% of FNDs is enough to track PLA nanoparticles in tissue. Apart from what we have shown here, FNDs may also be used as a tool to track the polymer degradation in real time by quantum sensing. (39)

Intracellular Location of Nanoparticles

In order to further follow up on intracellular trafficking routes of particles, endosomes and lysosomes of macrophages were marked by EEA1 antibody and LAMP-1 antibody (see Figures 5 and 6). These organelles were chosen because endosomes and lysosomes are the main organelles through which internalized nanoparticles pass. We determined the proportion of FNDs and PLA-FNDs that colocalized with early endosomes EEA1 marked at 0, 6, and 24 h (see Figure 7). We found no statistically significant difference between 0 and 24 h for both FNDs and PLA-FNDs. For the FND group, the colocalization proportion with early endosomes was 19.43%, 22.79%, and 24.17% at 0 h, 6 h, and 24 h of incubation. FNDs are aggregated more at 6 h as a result of intracellular uptake. These results suggest that only a small part of FNDs were internalized in early endosomes, and most FNDs were already in the cytoplasm or late endosomes or lysosomes. The colocalization proportion of FNDs internalized in late endosomes or LMAP-1 marked lysosomes increased significantly over time from 27.27% for 0 h incubation to 44.32% for 6 h incubation and 51.07% for 24 h incubation. This indicates that the early endosomes where most FNDs were internalized have become late endosomes or lysosomes. It is possible that some FNDs escaped from endosomes to the cytoplasm and were translocated into lysosomes as the uptake time increased. It is also possible that a small part of the FNDs escaped from endosomes into the cytoplasm but did not translocate into lysosomes. Similar cases have been reported in some studies. (40,41)

Figure 5

Figure 5. Colocalization of PLA and PLA-FNDs with early endosome compartments labeled by EEA1 antibody after 0, 6, and 24 h of incubation (red: FNDs, green: early endosomes, blue: nucleus). Merged images are shown. All data were analyzed from at least 50 cells.

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Figure 6

Figure 6. Colocalization of FNDs and PLA-FNDs with lysosome compartments labeled by LAMP-1 antibodies after 0, 6, and 24 h of incubation (red: FNDs, green: late endosomes and lysosomes, blue: nucleus). Merged images are shown. All data were obtained from at least 50 cells.

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Figure 7

Figure 7. Quantitative colocalization analysis of ingested FNDs and PLA-FNDs with endosomes and lysosomes from confocal microscopy images. All data are shown as mean ± standard deviation for a total of 50 cells at least. (A) Quantitative analysis of FNDs, PLA-FND colocalization with early endosomes labeled with EEA1. The results indicate no statistically significant difference between the different groups. (B) Quantitative analysis of FNDs, PLA-FND colocalization with late endosomes and lysosomes labeled with LAMP-1. The results indicated statistically significant differences between FNDs or PLA-FNDs.

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For the PLA-FND group, the colocalization proportion of PLA-FNDs internalized at early endosomes is similar to FNDs. But, unlike FNDs, the proportion of PLA-FNDs colocalized with late endosomes or lysosomes, and LMAP-1 marked decreases significantly with incubation time. For 0 h incubation, internalization of PLA-FNDs was 26.52% in early endosomes, and internalization of PLA-FNDs was 56.75% in late endosomes and lysosomes. With the increase of incubation time, the proportion of PLA-FNDs internalized at early endosomes was still low at 6 h and 24 h incubation, reaching 22.36 and 19.63%, respectively. However, internalization of PLA-FNDs with late endosomes and lysosomes decreased to 42.35 and 34.56%. This result suggested that the early endosomes where PLA-FNDs were internalized had become late endosomes or lysosomes in the early uptake. It is likely that endosome or lysosome membranes were disrupted as uptake time increased since the endosome and lysosome environment is acidic with pH 5.0. PLA could be degraded in this environment, and then fragments will interact directly with the endosomal and lysosomal membrane and disrupt the membrane. (42)

Recording FNDs and PLA-FND Trajectories in Living Cells

In order to further explore the movement of particles inside cells, trajectories were recorded. Figure 8 shows the mean diffusion coefficient of 8 h trajectories on the xy-plane. We found diffusion coefficients much lower than those of FBS-coated 70 nm FNDs and 120 nm FNDs in HeLa cells. The behavior of PLA-FNDs 01 did not differ from those of the three FNDs tested, and PLA-FNDs 02 and 03 did not differ from FNDs 01 and 02 but differed significantly from FND 03, possibly because of differences between particles or the extent of aggregation of internalized particles. In addition, the movement trajectories of PLA-FNDs in the xy-plane did not change significantly over time (Figure S3 in Supporting Information). However, the volume that particles explored decreased more rapidly in FND-PLA compared with FNDs (see Figure S4 in the Supporting Information). Additionally, system drift could have a certain localization error, but there is not a massive impact on trajectories based on previous work. (43) To summarize, the trajectory of a particle could be affected by many factors, and cells respond to different particle shapes and sizes, which influence trajectories.

Figure 8

Figure 8. Mean diffusion coefficient during 8 h measurements in the xy-plane. Three FNDs and three PLA-FNDs were tested. Black dotted lines indicate the median diffusion coefficient of FBS-coated 70 and 120 nm FNDs in HeLa cells from previous work (43) (different bare particles are numbered as FND 01, FND 02, and FND 03, while PLA-FND 01, PLA-FND 02, and PLA-FND 03 indicate FNDs that are in PLA particles).

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Conclusions

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FNDs offer stable fluorescence and do not bleach or blink. As a result, they are attractive labels for long-term imaging and long-term tracking. Here, we have shown that these particles can be used to visualize, where polymer nanoparticles are transported within cells or tissues. More specifically, we were able to follow the uptake route in cells. We also quantified the number and volume of particles in the cell and evaluated the colocalization of the particles and organelles. In addition, PLA-FNDs have diffusion coefficients lower than FNDs. This indicates that either FNDs or aggregates or PLA-FNDs are transported differently. In tissues, we were able to observe PLA-FNDs using two-photon microscopy. While this work was limited to studying PLA fluorescent nanodiamonds, they can be applied to other polymer particles in the same fashion.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.3c01452.

  • Protocols for nanodiamond tracking; characterization of PLA-FNDs, including SEM, DLS, confocal images; tracking results; XTT assay results to confirm the biocompatibility of particles; and details on quantitative image analysis (PDF)

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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  • Corresponding Author
  • Authors
    • Runrun Li - Department of Biomedical Engineering, Groningen University, University Medical Center Groningen, Antonius Deusinglaan 1, 9713AV Groningen, The Netherlands
    • Thea A. Vedelaar - Department of Biomedical Engineering, Groningen University, University Medical Center Groningen, Antonius Deusinglaan 1, 9713AV Groningen, The Netherlands
    • Alina Sigaeva - Department of Biomedical Engineering, Groningen University, University Medical Center Groningen, Antonius Deusinglaan 1, 9713AV Groningen, The Netherlands
    • Yue Zhang - Department of Biomedical Engineering, Groningen University, University Medical Center Groningen, Antonius Deusinglaan 1, 9713AV Groningen, The Netherlands
    • Kaiqi Wu - Department of Biomedical Engineering, Groningen University, University Medical Center Groningen, Antonius Deusinglaan 1, 9713AV Groningen, The Netherlands
    • Hui Wang - Zernike Institute for Advanced Materials, Groningen University, Nijenborgh 4, 9747 AG Groningen, The Netherlands
    • Xixi Wu - Department of Biomedical Engineering, Groningen University, University Medical Center Groningen, Antonius Deusinglaan 1, 9713AV Groningen, The Netherlands;  Zernike Institute for Advanced Materials, Groningen University, Nijenborgh 4, 9747 AG Groningen, The Netherlands
    • Peter Olinga - Department of Pharmaceutical Technology and Biopharmacy, Groningen University, University Medical Center Groningen, Antonius Deusinglaan 1, 9713AV Groningen, The Netherlands
    • Małgorzata K. Wlodarzyk-Biegun - Zernike Institute for Advanced Materials, Groningen University, Nijenborgh 4, 9747 AG Groningen, The Netherlands;  Biotechnology Centre, The Silesian University of Technology, Krzywoustego 8, 44-100 Gliwice, Poland
  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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R.L. acknowledges financial support from the Chinese government via a CSC scholarship (No. 201906220229). R.S. is grateful for financial support from the NWO via a VIDI grant (016.Vidi.189.002) and MW via a VENI grant (VI.Veni.192.148).

References

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    There is no corresponding record for this reference.
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    Feng, X.; Lv, F.; Liu, L.; Tang, H.; Xing, C.; Yang, Q.; Wang, S. Conjugated polymer nanoparticles for drug delivery and imaging. ACS Appl. Mater. Interfaces 2010, 2 (8), 2429– 2435,  DOI: 10.1021/am100435k
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    Conjugated Polymer Nanoparticles for Drug Delivery and Imaging
    Feng, Xuli; Lv, Fengting; Liu, Libing; Tang, Hongwei; Xing, Chengfen; Yang, Qiong; Wang, Shu
    ACS Applied Materials & Interfaces (2010), 2 (8), 2429-2435CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)
    We prepd. a new conjugated polymer nanoparticle with the size of about 50 nm that is prepd. by electrostatic assembly of cationic conjugated polymer PFO and anionic poly(L-glutamic acid) conjugated with anticancer drug doxorubicin (PFO/PG-Dox). The PFO exhibits good fluorescence quantum yield, photostability, and little cytotoxicity to meet the essential requests for cell imaging. In PFO/PG-Dox nanoparticles, the fluorescence of PFO is highly quenched by Dox by electron transfer mechanism, and thus the PFO is in the fluorescence "turn-off' state. After PFO/PG-Dox nanoparticles are exposed to carboxypeptidase or are taken up by cancer cells, the poly(L-glutamic acid) is hydrolyzed to release the Dox, inducing the activation of PFO fluorescence to "turn-on" state. This multifunctional nanoparticle system can deliver Dox to targeted cancer cells and monitor the Dox release based on fluorescence "turn-on" signal of PFO, which concurrently images the cancer cells. The present work opens the door for new functional studies of conjugated polymer in simultaneous imaging and disease therapeutics.
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  3. 3
    Forier, K.; Raemdonck, K.; De Smedt, S. C.; Demeester, J.; Coenye, T.; Braeckmans, K. Lipid and polymer nanoparticles for drug delivery to bacterial biofilms. J. Controlled Release 2014, 190, 607– 623,  DOI: 10.1016/j.jconrel.2014.03.055
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    3
    Lipid and polymer nanoparticles for drug delivery to bacterial biofilms
    Forier, Katrien; Raemdonck, Koen; De Smedt, Stefaan C.; Demeester, Jo; Coenye, Tom; Braeckmans, Kevin
    Journal of Controlled Release (2014), 190 (), 607-623CODEN: JCREEC; ISSN:0168-3659. (Elsevier B.V.)
    A review. Biofilms are matrix-enclosed communities of bacteria that show increased antibiotic resistance and the capability to evade the immune system. They can cause recalcitrant infections which cannot be cured with classical antibiotic therapy. Drug delivery by lipid or polymer nanoparticles is considered a promising strategy for overcoming biofilm resistance. These particles are able to improve the delivery of antibiotics to the bacterial cells, thereby increasing the efficacy of the treatment. In this review we give an overview of the types of polymer and lipid nanoparticles that have been developed for this purpose. The antimicrobial activity of nanoparticle encapsulated antibiotics compared to the activity of the free antibiotic is discussed in detail. In addn., targeting and triggered drug release strategies to further improve the antimicrobial activity are reviewed. Finally, ample attention is given to advanced microscopy methods that shed light on the behavior of nanoparticles inside biofilms, allowing further optimization of the nanoformulations. Lipid and polymer nanoparticles were found to increase the antimicrobial efficacy in many cases. Strategies such as the use of fusogenic liposomes, targeting of the nanoparticles and triggered release of the antimicrobial agent ensured the delivery of the antimicrobial agent in close proximity of the bacterial cells, maximizing the exposure of the biofilm to the antimicrobial agent. The majority of the discussed papers still present data on the in vitro anti-biofilm activity of nanoformulations, indicating that there is an urgent need for more in vivo studies in this field.
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  4. 4
    Sharifi, S.; Behzadi, S.; Laurent, S.; Forrest, M. L.; Stroeve, P.; Mahmoudi, M. Toxicity of nanomaterials. Chem. Soc. Rev. 2012, 41 (6), 2323– 2343,  DOI: 10.1039/C1CS15188F
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    Toxicity of nanomaterials
    Sharifi, Shahriar; Behzadi, Shahed; Laurent, Sophie; Laird Forrest, M.; Stroeve, Pieter; Mahmoudi, Morteza
    Chemical Society Reviews (2012), 41 (6), 2323-2343CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
    A review. Nanoscience has matured significantly during the last decade as it has transitioned from bench top science to applied technol. Presently, nanomaterials are used in a wide variety of com. products such as electronic components, sports equipment, sun creams and biomedical applications. There are few studies of the long-term consequences of nanoparticles on human health, but governmental agencies, including the United States National Institute for Occupational Safety and Health and Japan's Ministry of Health, have recently raised the question of whether seemingly innocuous materials such as carbon-based nanotubes should be treated with the same caution afforded known carcinogens such as asbestos. Since nanomaterials are increasing a part of everyday consumer products, manufg. processes, and medical products, it is imperative that both workers and end-users be protected from inhalation of potentially toxic NPs. It also suggests that NPs may need to be sequestered into products so that the NPs are not released into the atm. during the product's life or during recycling. Further, non-inhalation routes of NP absorption, including dermal and medical injectables, must be studied in order to understand possible toxic effects. Fewer studies to date have addressed whether the body can eventually eliminate nanomaterials to prevent particle build-up in tissues or organs. This crit. review discusses the biophysicochem. properties of various nanomaterials with emphasis on currently available toxicol. data and methodologies for evaluating nanoparticle toxicity (286 refs.).
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  5. 5
    Paul, M. B.; Stock, V.; Cara-Carmona, J.; Lisicki, E.; Shopova, S.; Fessard, V.; Braeuning, A.; Sieg, H.; Böhmert, L. Micro-and nanoplastics–current state of knowledge with the focus on oral uptake and toxicity. Nanoscale Adv. 2020, 2 (10), 4350– 4367,  DOI: 10.1039/D0NA00539H
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    5
    Micro- and nanoplastics - current state of knowledge with the focus on oral uptake and toxicity
    Paul, Maxi B.; Stock, Valerie; Cara-Carmona, Julia; Lisicki, Elisa; Shopova, Sofiya; Fessard, Valerie; Braeuning, Albert; Sieg, Holger; Boehmert, Linda
    Nanoscale Advances (2020), 2 (10), 4350-4367CODEN: NAADAI; ISSN:2516-0230. (Royal Society of Chemistry)
    A review. The prodn. and use of plastics has constantly increased over the last 30 years. Over one third of the plastics is used in disposables, which are discarded within three years of their prodn. Despite efforts towards recycling, a substantial vol. of debris has accumulated in the environment and is slowly degraded to micro- and nanoplastics by weathering and aging. It has recently been discovered that these small particles can enter the food chain, as for example demonstrated by the detection of microplastic particles in honey, beer, salt, sea food and recently in mineral water. Human exposure has further been documented by the detection of plastic microparticles in human feces. Potential toxic consequences of oral exposure to small plastic particles are discussed. Due to lacking data concerning exposure, biodistribution and related effects, the risk assessment of micro- and nanoplastics is still not possible. This review focuses on the oral uptake of plastic and polymer micro- and nanoparticles. Oral exposure, particle fate, changes of particle properties during ingestion and gastrointestinal digestion, and uptake and transport at the intestinal epithelium are reviewed in detail. Moreover, the interaction with intestinal and liver cells and possibly resulting toxicity are highlighted.
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  6. 6
    Mariano, S.; Tacconi, S.; Fidaleo, M.; Rossi, M.; Dini, L. Micro and nanoplastics identification: classic methods and innovative detection techniques. Front. Toxicol. 2021, 3, 636640  DOI: 10.3389/ftox.2021.636640
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    6
    Micro and Nanoplastics Identification: Classic Methods and Innovative Detection Techniques
    Mariano Stefania; Tacconi Stefano; Fidaleo Marco; Dini Luciana; Rossi Marco; Rossi Marco; Dini Luciana; Rossi Marco; Dini Luciana
    Frontiers in toxicology (2021), 3 (), 636640 ISSN:.
    Micro and nanoplastics are fragments with dimensions less than a millimeter invading all terrestrial and marine environments. They have become a major global environmental issue in recent decades and, indeed, recent scientific studies have highlighted the presence of these fragments all over the world even in environments that were thought to be unspoiled. Analysis of micro/nanoplastics in isolated samples from abiotic and biotic environmental matrices has become increasingly common. Hence, the need to find valid techniques to identify these micro and nano-sized particles. In this review, we discuss the current and potential identification methods used in microplastic analyses along with their advantages and limitations. We discuss the most suitable techniques currently available, from physical to chemical ones, as well as the challenges to enhance the existing methods and develop new ones. Microscopical techniques (i.e., dissect, polarized, fluorescence, scanning electron, and atomic force microscopy) are one of the most used identification methods for micro/nanoplastics, but they have the limitation to produce incomplete results in analyses of small particles. At present, the combination with chemical analysis (i.e., spectroscopy) overcome this limit together with recently introduced alternative approaches. For example, holographic imaging in microscope configuration images microplastics directly in unfiltered water, thus discriminating microplastics from diatoms and differentiates different sizes, shapes, and plastic types. The development of new analytical instruments coupled with each other or with conventional and innovative microscopy could solve the current problems in the identification of micro/nanoplastics.
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  7. 7
    Li, G.; Yang, Z.; Pei, Z.; Li, Y.; Yang, R.; Liang, Y.; Zhang, Q.; Jiang, G. Single-particle analysis of micro/nanoplastics by SEM-Raman technique. Talanta 2022, 249, 123701  DOI: 10.1016/j.talanta.2022.123701
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    7
    Single-particle analysis of micro/nanoplastics by SEM-Raman technique
    Li, Gang; Yang, Zhiruo; Pei, Zhiguo; Li, Yingming; Yang, Ruiqiang; Liang, Yong; Zhang, Qinghua; Jiang, Guibin
    Talanta (2022), 249 (), 123701CODEN: TLNTA2; ISSN:0039-9140. (Elsevier B.V.)
    Micro/nanoplastics (MNPs) have received global concern due to their widespread contamination, ingestion in organisms, and the ability to cross the biol. barrier. Although MNPs have been detected in a variety of ecosystems, the identification of single MNPs remains an unsolved challenge. Herein, for the first time, scanning electron microscope (SEM) coupled with surface-enhanced Raman spectroscopy (SERS), which combined the advantages of ultrahigh spatial resoln. of SEM and structural fingerprint of Raman spectroscopy, was proposed to identify MNPs at single-particle level. Under the optimum conditions, the polystyrene (PS) MNPs with sizes of 500 nm and 1μm were identified by the image of SEM and fingerprint peaks of Raman spectroscopy. Addnl., the applicability of the method in different sample matrixes and for other types of MNPs such as poly-Me methacrylate (PMMA) with the sizes of 300 nm, 1μm were validated. This method is simple, rapid and effective and is likely to provide an essential tool to identify other micro/nanoparticles in addn. to MNPs.
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  8. 8
    Shim, W. J.; Song, Y. K.; Hong, S. H.; Jang, M. Identification and quantification of microplastics using Nile Red staining. Mar. Pollut. Bull. 2016, 113 (1–2), 469– 476,  DOI: 10.1016/j.marpolbul.2016.10.049
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    8
    Identification and quantification of microplastics using Nile Red staining
    Shim, Won Joon; Song, Young Kyoung; Hong, Sang Hee; Jang, Mi
    Marine Pollution Bulletin (2016), 113 (1-2), 469-476CODEN: MPNBAZ; ISSN:0025-326X. (Elsevier Ltd.)
    We investigated the applicability of Nile Red (NR), a fluorescent dye, for microplastic anal., and detd. the optimal staining conditions. Five mg/L NR soln. in n-hexane effectively stained plastics, and they were easily recognized in green fluorescence. The NR staining method was successfully applied to micro-sized polyethylene, polypropylene, polystyrene, polycarbonate, polyurethane, and poly(ethylene-vinyl acetate), except for polyvinylchloride, polyamide and polyester. The recovery rate of polyethylene (100-300 μm) spiked to pretreated natural sand was 98% in the NR stating method, which was not significantly (p < 0.05) different with FT-IR identification. The NR staining method was suitable for discriminating fragmented polypropylene particles from large nos. of sand particles in lab. weathering test samples. The method is straightforward and quick for identifying and quantifying polymer particles in the lab. controlled samples. Further studies, however, are necessary to investigate the application of NR staining to field samples with org. remnants.
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  9. 9
    Lv, L.; He, L.; Jiang, S.; Chen, J.; Zhou, C.; Qu, J.; Lu, Y.; Hong, P.; Sun, S.; Li, C. In situ surface-enhanced Raman spectroscopy for detecting microplastics and nanoplastics in aquatic environments. Sci. Total Environ. 2020, 728, 138449  DOI: 10.1016/j.scitotenv.2020.138449
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    9
    In situ surface-enhanced Raman spectroscopy for detecting microplastics and nanoplastics in aquatic environments
    Lv, Lulu; He, Lei; Jiang, Shiqi; Chen, Jinjun; Zhou, Chunxia; Qu, Junhao; Lu, Yuqin; Hong, Pengzhi; Sun, Shengli; Li, Chengyong
    Science of the Total Environment (2020), 728 (), 138449CODEN: STENDL; ISSN:0048-9697. (Elsevier B.V.)
    Micro- and nano-plastics detection in environmental matrixes, particularly in-situ plastic particles in aquatic environments, still is challenged due to limitations of existing methods, instruments, and plastic particle sizes. This work evaluated the potential of surface-enhanced Raman spectroscopy to analyze micro- and nano-plastics. Conditions for different tests (sample:Ag colloid vol. ratio, NaCl and sample concns.) were studied to assess micro- and nano-plastics (polystyrene [PS], polyethylene [PE], polypropylene [PP]) in pure water and seawater. A SERS-based method which uses Ag colloid as the active substrate was developed to qual. analyze micro- and nano-plastics in aquatic environments. Micro- and nano-plastic particle sizes were 100 and 500 nm and 10μm. Micro- and nano-plastics Raman signals in pure water and seawater both showed good enhancement efficiency. The optimal enhancement factor was 4 x 104. The SERS-based detection method overcame limitations of micro- and nano-plastics in liqs. and detects 100 nm plastics down to 40μg/mL. It provides greater possibilities for future rapid micro- and nano-plastics detection in aquatic environments.
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  10. 10
    Vaijayanthimala, V.; Cheng, P. Y.; Yeh, S. H.; Liu, K. K.; Hsiao, C. H.; Chao, J. I.; Chang, H. C. The long-term stability and biocompatibility of fluorescent nanodiamond as an in vivo contrast agent. Biomaterials 2012, 33 (31), 7794– 7802,  DOI: 10.1016/j.biomaterials.2012.06.084
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    10
    The long-term stability and biocompatibility of fluorescent nanodiamond as an in vivo contrast agent
    Vaijayanthimala, V.; Cheng, Po-Yun; Yeh, Shih-Hua; Liu, Kuang-Kai; Hsiao, Cheng-Hsiang; Chao, Jui-I.; Chang, Huan-Cheng
    Biomaterials (2012), 33 (31), 7794-7802CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)
    Nanocarbon is a promising type of biomaterial for diagnostic and therapeutic applications. Fluorescent nanodiamond (FND) contg. nitrogen-vacancy centers as built-in fluorophores is a new addn. to the nanocarbon family. Here, we study the long-term stability and biocompatibility of 100-nm FNDs in rats through i.p. injection over 5 mo and develop the potential application of this biomaterial for sentinel lymph node mapping in a mouse model. From both in vivo and ex vivo fluorescence imaging as well as transmission electron microscopy, we found that the intradermally administered FND particles can be drained from the injection sites by macrophages and selectively accumulated in the axillary lymph nodes of the treated mice. Our measurements of water consumption, fodder consumption, body wt., and organ index showed no significant difference between control and FND-treated groups of the rats. Histopathol. anal. of various tissues and organs indicated that FNDs are non-toxic even when a large quantity, up to 75 mg/kg body wt., of the particles was administered i.p. to the living animals. With the properties of wide-ranging biocompatibility and perfect chem. and photophys. stability, FND is well suited for use as a contrast agent for long-term in vivo imaging.
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  11. 11
    Vaijayanthimala, V.; Tzeng, Y. K.; Chang, H. C.; Li, C. L. The biocompatibility of fluorescent nanodiamonds and their mechanism of cellular uptake. Nanotechnology 2009, 20 (42), 425103  DOI: 10.1088/0957-4484/20/42/425103
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    11
    The biocompatibility of fluorescent nanodiamonds and their mechanism of cellular uptake
    Vaijayanthimala, Vairakkannu; Tzeng, Yan-Kai; Chang, Huan-Cheng; Li, Chung-Leung
    Nanotechnology (2009), 20 (42), 425103/1-425103/9CODEN: NNOTER; ISSN:1361-6528. (Institute of Physics Publishing)
    The labeling of cells with fluorescent nanoparticles is promising for various biomedical applications. The objective of this study is to evaluate the biocompatibility and the mechanism of the cellular uptake of fluorescent nanodiamonds (FNDs) in cancer cells (HeLa) and pre-adipocytes (3T3-L1). With flow cytometry and the use of a battery of metabolic and cytoskeletal inhibitors, we found that the mechanism of the FND uptake in both cells is by energy-dependent clathrin-mediated endocytosis. In addn., the surface charge of FND influences its cellular uptake, as the uptake of poly-L-lysine-coated FNDs is better than that of oxidative-acid-purified FNDs at the same concn. in regular medium with or without serum. We also confirm that the proliferative potential of FND-treated and untreated cells does not exhibit any significant differences when measured at bulk cultures, and more stringently at clonal cell d. Further biocompatibility studies indicate that the in vitro differentiation of 3T3-L1 pre-adipocytes and 489-2 osteoprogenitors is not affected by the FND treatment. Our results show that FNDs are biocompatible and ideal candidates for potential applications in human stem cell research.
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  12. 12
    Hsu, T. C.; Liu, K. K.; Chang, H. C.; Hwang, E.; Chao, J. I. Labeling of neuronal differentiation and neuron cells with biocompatible fluorescent nanodiamonds. Sci. Rep. 2014, 4 (1), 5004  DOI: 10.1038/srep05004
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    12
    Labeling of neuronal differentiation and neuron cells with biocompatible fluorescent nanodiamonds
    Hsu, Tzu-Chia; Liu, Kuang-Kai; Chang, Huan-Cheng; Hwang, Eric; Chao, Jui-I.
    Scientific Reports (2014), 4 (), 5004CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
    Nanodiamond is a promising carbon nanomaterial developed for biomedical applications. Here, we show fluorescent nanodiamond (FND) with the biocompatible properties that can be used for the labeling and tracking of neuronal differentiation and neuron cells derived from embryonal carcinoma stem (ECS) cells. The fluorescence intensities of FNDs were increased by treatment with FNDs in both the mouse P19 and human NT2/D1 ECS cells. FNDs were taken into ECS cells; however, FNDs did not alter the cellular morphol. and growth ability. Moreover, FNDs did not change the protein expression of stem cell marker SSEA-1 of ECS cells. The neuronal differentiation of ECS cells could be induced by retinoic acid (RA). Interestingly, FNDs did not affect on the morphol. alteration, cytotoxicity and apoptosis during the neuronal differentiation. Besides, FNDs did not alter the cell viability and the expression of neuron-specific marker β-III-tubulin in these differentiated neuron cells. The existence of FNDs in the neuron cells can be identified by confocal microscopy and flow cytometry. Together, FND is a biocompatible and readily detectable nanomaterial for the labeling and tracking of neuronal differentiation process and neuron cells from stem cells.
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  13. 13
    van der Laan, K.; Hasani, M.; Zheng, T.; Schirhagl, R. Nanodiamonds for in vivo applications. Small 2018, 14 (19), 1703838  DOI: 10.1002/smll.201703838
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    There is no corresponding record for this reference.
  14. 14
    Haziza, S.; Mohan, N.; Loe-Mie, Y.; Lepagnol-Bestel, A. M.; Massou, S.; Adam, M. P.; Le, X. L.; Viard, J.; Plancon, C.; Daudin, R.; Koebel, P. Fluorescent nanodiamond tracking reveals intraneuronal transport abnormalities induced by brain-disease-related genetic risk factors. Nat. Nanotechnol. 2017, 12 (4), 322– 328,  DOI: 10.1038/nnano.2016.260
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    14
    Fluorescent nanodiamond tracking reveals intraneuronal transport abnormalities induced by brain-disease-related genetic risk factors
    Haziza, Simon; Mohan, Nitin; Loe-Mie, Yann; Lepagnol-Bestel, Aude-Marie; Massou, Sophie; Adam, Marie-Pierre; Le, Xuan Loc; Viard, Julia; Plancon, Christine; Daudin, Rachel; Koebel, Pascale; Dorard, Emilie; Rose, Christiane; Hsieh, Feng-Jen; Wu, Chih-Che; Potier, Brigitte; Herault, Yann; Sala, Carlo; Corvin, Aiden; Allinquant, Bernadette; Chang, Huan-Cheng; Treussart, Francois; Simonneau, Michel
    Nature Nanotechnology (2017), 12 (4), 322-328CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
    Brain diseases such as autism and Alzheimer's disease (each inflicting >1% of the world population) involve a large network of genes displaying subtle changes in their expression. Abnormalities in intraneuronal transport have been linked to genetic risk factors found in patients, suggesting the relevance of measuring this key biol. process. However, current techniques are not sensitive enough to detect minor abnormalities. Here the authors report a sensitive method to measure the changes in intraneuronal transport induced by brain-disease-related genetic risk factors using fluorescent nanodiamonds (FNDs). The authors show that the high brightness, photostability and absence of cytotoxicity allow FNDs to be tracked inside the branches of dissocd. neurons with a spatial resoln. of 12 nm and a temporal resoln. of 50 ms. As proof of principle, the authors applied the FND tracking assay on two transgenic mouse lines that mimic the slight changes in protein concn. (∼30%) found in the brains of patients. In both cases, the authors show that the FND assay is sufficiently sensitive to detect these changes.
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  15. 15
    Wu, T. J.; Tzeng, Y. K.; Chang, W. W.; Cheng, C. A.; Kuo, Y.; Chien, C. H.; Chang, H. C.; Yu, J. Tracking the engraftment and regenerative capabilities of transplanted lung stem cells using fluorescent nanodiamonds. Nat. Nanotechnol. 2013, 8 (9), 682– 689,  DOI: 10.1038/nnano.2013.147
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    15
    Tracking the engraftment and regenerative capabilities of transplanted lung stem cells using fluorescent nanodiamonds
    Wu, Tsai-Jung; Tzeng, Yan-Kai; Chang, Wei-Wei; Cheng, Chi-An; Kuo, Yung; Chien, Chin-Hsiang; Chang, Huan-Cheng; Yu, John
    Nature Nanotechnology (2013), 8 (9), 682-689CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
    Lung stem/progenitor cells are potentially useful for regenerative therapy, for example in repairing damaged or lost lung tissue in patients. Several optical imaging methods and probes have been used to track how stem cells incorporate and regenerate themselves in vivo over time. However, these approaches are limited by photobleaching, toxicity and interference from background tissue autofluorescence. Here we show that fluorescent nanodiamonds, in combination with fluorescence-activated cell sorting, fluorescence lifetime imaging microscopy and immunostaining, can identify transplanted CD45-CD54+CD157+ lung stem/progenitor cells in vivo, and track their engraftment and regenerative capabilities with single-cell resoln. Fluorescent nanodiamond labeling did not eliminate the cells' properties of self-renewal and differentiation into type I and type II pneumocytes. Time-gated fluorescence imaging of tissue sections of naphthalene-injured mice indicates that the fluorescent nanodiamond-labeled lung stem/progenitor cells preferentially reside at terminal bronchioles of the lungs for 7 days after i.v. transplantation.
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  16. 16
    Sigaeva, A.; Hochstetter, A.; Bouyim, S.; Chipaux, M.; Stejfova, M.; Cigler, P.; Schirhagl, R. Single-Particle Tracking and Trajectory Analysis of Fluorescent Nanodiamonds in Cell-Free Environment and Live Cells. Small 2022, 18 (39), 2201395  DOI: 10.1002/smll.202201395
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    16
    Single-Particle Tracking and Trajectory Analysis of Fluorescent Nanodiamonds in Cell-Free Environment and Live Cells
    Sigaeva, Alina; Hochstetter, Axel; Bouyim, Sighom; Chipaux, Mayeul; Stejfova, Miroslava; Cigler, Petr; Schirhagl, Romana
    Small (2022), 18 (39), 2201395CODEN: SMALBC; ISSN:1613-6810. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Diamond magnetometry can provide new insights on the prodn. of free radicals inside live cells due to its high sensitivity and spatial resoln. However, the measurements often lack intracellular context for the recorded signal. In this paper, the possible use of single-particle tracking and trajectory anal. of fluorescent nanodiamonds (FNDs) to bridge that gap is explored. It starts with simulating a set of different possible scenarios of a particles movement, reflecting different modes of motion, degrees of confinement, as well as shapes and sizes of that confinement. Then, the insights from the anal. of the simulated trajectories are applied to describe the movement of FNDs in glycerol solns. It is shown that the measurements are in good agreement with the previously reported findings and that trajectory anal. 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.
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  17. 17
    Hui, Y. Y.; Hsiao, W. W. W.; Haziza, S.; Simonneau, M.; Treussart, F.; Chang, H. C. Single particle tracking of fluorescent nanodiamonds in cells and organisms. Curr. Opin. Solid State Mater. Sci. 2017, 21 (1), 35– 42,  DOI: 10.1016/j.cossms.2016.04.002
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    Single particle tracking of fluorescent nanodiamonds in cells and organisms
    Hui, Yuen Yung; Hsiao, Wesley Wei-Wen; Haziza, Simon; Simonneau, Michel; Treussart, Francois; Chang, Huan-Cheng
    Current Opinion in Solid State & Materials Science (2017), 21 (1), 35-42CODEN: COSSFX; ISSN:1359-0286. (Elsevier Ltd.)
    Ever since the discovery of fullerenes in 1985, nanocarbon has demonstrated a wide range of applications in various areas of science and engineering. Compared with metal, oxide, and semiconductor nanoparticles, the carbon-based nanomaterials have distinct advantages in both biotechnol. and biomedical applications due to their inherent biocompatibility. Fluorescent nanodiamond (FND) joined the nanocarbon family in 2005. It was initially developed as a contrast agent for bioimaging because it can emit bright red photoluminescence from neg. charged nitrogen-vacancy centers built in the diamond matrix. A notable application of this technol. is to study the cytoplasmic dynamics of living cells by tracking single bioconjugated FNDs in intracellular medium. This article provides a crit. review on recent advances and developments of such single particle tracking (SPT) research. It summarizes SPT and related studies of FNDs in cells (such as cancer cell lines) and organisms (including zebrafish embryos, fruit fly embryos, whole nematodes, and mice) using assorted imaging techniques.
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  18. 18
    Ghanimi Fard, M.; Khabir, Z.; Reineck, P.; Cordina, N. M.; Abe, H.; Ohshima, T.; Dalal, S.; Gibson, B. C.; Packer, N. H.; Parker, L. M. Targeting cell surface glycans with lectin-coated fluorescent nanodiamonds. Nanoscale Adv. 2022, 4 (6), 1551– 1564,  DOI: 10.1039/D2NA00036A
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    18
    Targeting cell surface glycans with lectin-coated fluorescent nanodiamonds
    Ghanimi Fard, Mina; Khabir, Zahra; Reineck, Philipp; Cordina, Nicole M.; Abe, Hiroshi; Ohshima, Takeshi; Dalal, Sagar; Gibson, Brant C.; Packer, Nicolle H.; Parker, Lindsay M.
    Nanoscale Advances (2022), 4 (6), 1551-1564CODEN: NAADAI; ISSN:2516-0230. (Royal Society of Chemistry)
    Glycosylation is arguably the most important functional post-translational modification in brain cells and abnormal cell surface glycan expression has been assocd. with neurol. diseases and brain cancers. In this study we developed a novel method for uptake of fluorescent nanodiamonds (FND), carbon-based nanoparticles with low toxicity and easily modifiable surfaces, into brain cell subtypes by targeting their glycan receptors with carbohydrate-binding lectins. Lectins facilitated uptake of 120 nm FND with nitrogen-vacancy centers in three types of brain cells - U87-MG astrocytes, PC12 neurons and BV-2 microglia cells. The nanodiamond/lectin complexes used in this study target glycans that have been described to be altered in brain diseases including sialic acid glycans via wheat (Triticum aestivum) germ agglutinin (WGA), high mannose glycans via tomato (Lycopersicon esculentum) lectin (TL) and core fucosylated glycans via Aleuria aurantia lectin (AAL). The lectin conjugated nanodiamonds were taken up differently by the various brain cell types with fucose binding AAL/FNDs taken up preferentially by glioblastoma phenotype astrocyte cells (U87-MG), sialic acid binding WGA/FNDs by neuronal phenotype cells (PC12) and high mannose binding TL/FNDs by microglial cells (BV-2). With increasing recognition of glycans having a role in many diseases, the lectin bioconjugated nanodiamonds developed here are well suited for further investigation into theranostic applications.
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  19. 19
    Sharmin, R.; Nusantara, A. C.; Nie, L.; Wu, K.; Elias Llumbet, A.; Woudstra, W.; Mzyk, A.; Schirhagl, R. Intracellular Quantum Sensing of Free-Radical Generation Induced by Acetaminophen (APAP) in the Cytosol, in Mitochondria and the Nucleus of Macrophages. ACS Sens. 2022, 7 (11), 3326– 3334,  DOI: 10.1021/acssensors.2c01272
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    19
    Intracellular Quantum Sensing of Free-Radical Generation Induced by Acetaminophen (APAP) in the Cytosol, in Mitochondria and the Nucleus of Macrophages
    Sharmin, Rokshana; Nusantara, Anggrek C.; Nie, Linyan; Wu, Kaiqi; Elias Llumbet, Arturo; Woudstra, Willem; Mzyk, Aldona; Schirhagl, Romana
    ACS Sensors (2022), 7 (11), 3326-3334CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)
    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 resoln., these nanodiamonds were targeted to cellular locations, i.e., 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 prodn. in an organelle-specific manner, which helps predict and better understand cellular toxicity.
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    Terada, D.; Genjo, T.; Segawa, T. F.; Igarashi, R.; Shirakawa, M. Nanodiamonds for bioapplications–specific targeting strategies. Biochim. Biophys. Acta, Gen. Subj. 2020, 1864 (2), 129354  DOI: 10.1016/j.bbagen.2019.04.019
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    Nanodiamonds for bioapplications-specific targeting strategies
    Terada, Daiki; Genjo, Takuya; Segawa, Takuya F.; Igarashi, Ryuji; Shirakawa, Masahiro
    Biochimica et Biophysica Acta, General Subjects (2020), 1864 (2), 129354CODEN: BBGSB3; ISSN:0304-4165. (Elsevier B.V.)
    A review. Nanodiamonds (NDs) provide a unique multitasking system for drug delivery and fluorescent imaging in biol. environments. Owing to their quantum properties, NDs are expected to be employed as multifunctional probes in the future for the accurate visualization of biophys. parameters such as temp. and magnetic fields. However, the use of NDs for the selective targeting of the biomols. of interest within a complicated biol. system remains a challenge. One of the most promising solns. is the appropriate surface design of NDs based on org. chem. and biochem. The engineered NDs have high biocompatibility and dispersibility in a biol. environment and hence undergo cellular uptake through specific pathways. This review focuses on the selective targeting of NDs for biomedical and biophys. applications from the viewpoint of ND surface functionalizations and modifications. These pretreatments make possible the specific targeting of biomols. of interest on or in a cell by NDs via a designed biochem. route. The surface of NDs is covalently or noncovalently modified with silica, polymers, or biomols. to reshape them, control their size, and enhance the colloidal stability and biomol. selectivity toward the biomols. of interest. Electroporation, chem. treatment, injection, or endocytosis are the methods generally adopted to introduce NDs into living cells. The pathway, efficiency, and the cell viability depend on the selected method. In the biomedical field, the surface modification facilitates specific delivery of a drug, leading to a higher therapeutic efficacy. In biophys. applications, the surface modification paves the way for the accurate measurement of phys. parameters to gain a better understanding of various cell functions.
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  21. 21
    Hemelaar, S. R.; De Boer, P.; Chipaux, M.; Zuidema, W.; Hamoh, T.; Martinez, F. P.; Nagl, A.; Hoogenboom, J. P.; Giepmans, B. N. G.; Schirhagl, R. Nanodiamonds as multi-purpose labels for microscopy. Sci. Rep. 2017, 7 (1), 720  DOI: 10.1038/s41598-017-00797-2
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    Nanodiamonds as multi-purpose labels for microscopy
    Hemelaar S R; Chipaux M; Hamoh T; Martinez F Perona; Nagl A; Schirhagl R; de Boer P; Giepmans B N G; Zuidema W; Hoogenboom J P
    Scientific reports (2017), 7 (1), 720 ISSN:.
    Nanodiamonds containing fluorescent nitrogen-vacancy centers are increasingly attracting interest for use as a probe in biological microscopy. This interest stems from (i) strong resistance to photobleaching allowing prolonged fluorescence observation times; (ii) the possibility to excite fluorescence using a focused electron beam (cathodoluminescence; CL) for high-resolution localization; and (iii) the potential use for nanoscale sensing. For all these schemes, the development of versatile molecular labeling using relatively small diamonds is essential. Here, we show the direct targeting of a biological molecule with nanodiamonds as small as 70 nm using a streptavidin conjugation and standard antibody labelling approach. We also show internalization of 40 nm sized nanodiamonds. The fluorescence from the nanodiamonds survives osmium-fixation and plastic embedding making them suited for correlative light and electron microscopy. We show that CL can be observed from epon-embedded nanodiamonds, while surface-exposed nanoparticles also stand out in secondary electron (SE) signal due to the exceptionally high diamond SE yield. Finally, we demonstrate the magnetic read-out using fluorescence from diamonds prior to embedding. Thus, our results firmly establish nanodiamonds containing nitrogen-vacancy centers as unique, versatile probes for combining and correlating different types of microscopy, from fluorescence imaging and magnetometry to ultrastructural investigation using electron microscopy.
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  22. 22
    McGuinness, L. P.; Yan, Y.; Stacey, A.; Simpson, D. A.; Hall, L. T.; Maclaurin, D.; Prawer, S.; Mulvaney, P.; Wrachtrup, J.; Caruso, F.; Scholten, R. E.; Hollenberg, L. C. L. Quantum measurement and orientation tracking of fluorescent nanodiamonds inside living cells. Nat. Nanotechnol. 2011, 6 (6), 358– 363,  DOI: 10.1038/nnano.2011.64
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    Quantum measurement and orientation tracking of fluorescent nanodiamonds inside living cells
    McGuinness, L. P.; Yan, Y.; Stacey, A.; Simpson, D. A.; Hall, L. T.; MacLaurin, D.; Prawer, S.; Mulvaney, P.; Wrachtrup, J.; Caruso, F.; Scholten, R. E.; Hollenberg, L. C. L.
    Nature Nanotechnology (2011), 6 (6), 358-363CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
    Fluorescent particles are routinely used to probe biol. processes. The quantum properties of single spins within fluorescent particles have been explored in the field of nanoscale magnetometry, but not yet in biol. environments. Here, the authors demonstrate optically detected magnetic resonance of individual fluorescent nanodiamond nitrogen-vacancy centers inside living human HeLa cells, and measure their location, orientation, spin levels and spin coherence times with nanoscale precision. Quantum coherence was measured through Rabi and spin-echo sequences over long (>10 h) periods, and orientation was tracked with effective 1° angular precision over acquisition times of 89 ms. The quantum spin levels served as fingerprints, allowing individual centers with identical fluorescence to be identified and tracked simultaneously. Furthermore, monitoring decoherence rates in response to changes in the local environment may provide new information about intracellular processes. The expts. reported here demonstrate the viability of controlled single spin probes for nanomagnetometry in biol. systems, opening up a host of new possibilities for quantum-based imaging in the life sciences.
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    Neumann, P.; Jakobi, I.; Dolde, F.; Burk, C.; Reuter, R.; Waldherr, G.; Honert, J.; Wolf, T.; Brunner, A.; Shim, J. H.; Suter, D. High-precision nanoscale temperature sensing using single defects in diamond. Nano Lett. 2013, 13 (6), 2738– 2742,  DOI: 10.1021/nl401216y
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    High-Precision Nanoscale Temperature Sensing Using Single Defects in Diamond
    Neumann, P.; Jakobi, I.; Dolde, F.; Burk, C.; Reuter, R.; Waldherr, G.; Honert, J.; Wolf, T.; Brunner, A.; Shim, J. H.; Suter, D.; Sumiya, H.; Isoya, J.; Wrachtrup, J.
    Nano Letters (2013), 13 (6), 2738-2742CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
    Measuring local temp. with a spatial resoln. on the order of a few nanometers has a wide range of applications in the semiconductor industry and in material and life sciences. For example, probing temp. on the nanoscale with high precision can potentially be used to detect small, local temp. changes like those caused by chem. reactions or biochem. processes. However, precise nanoscale temp. measurements have not been realized so far owing to the lack of adequate probes. The authors exptl. demonstrate a novel nanoscale temp. sensing technique based on optically detected ESR in single at. defects in diamonds. These diamond sensor sizes range from a micrometer down to a few tens of nanometers. The authors achieve a temp. noise floor of 5 mK/Hz1/2 for single defects in bulk sensors. Using doped nano-diamonds as sensors, the temp. noise floor is 130 mK/Hz1/2 and accuracies down to 1 mK for nanocrystal sizes and therefore length scales of a few tens of nanometers. This combination of precision and position resoln., combined with the outstanding sensor photostability, should allow the measurement of the heat produced by chem. interactions involving a few or single mols. even in heterogeneous environments like cells.
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    Kucsko, G.; Maurer, P. C.; Yao, N. Y.; Kubo, M.; Noh, H. J.; Lo, P. K.; Park, H.; Lukin, M. D. Nanometre-scale thermometry in a living cell. Nature 2013, 500 (7460), 54– 58,  DOI: 10.1038/nature12373
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    Nanometre-scale thermometry in a living cell
    Kucsko, G.; Maurer, P. C.; Yao, N. Y.; Kubo, M.; Noh, H. J.; Lo, P. K.; Park, H.; Lukin, M. D.
    Nature (London, United Kingdom) (2013), 500 (7460), 54-58CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Sensitive probing of temp. variations on nanometer scales is an outstanding challenge in many areas of modern science and technol. In particular, a thermometer capable of subdegree temp. resoln. over a large range of temps. as well as integration within a living system could provide a powerful new tool in many areas of biol., phys. and chem. research. Possibilities range from the temp.-induced control of gene expression and tumor metab. to the cell-selective treatment of disease and the study of heat dissipation in integrated circuits. By combining local light-induced heat sources with sensitive nanoscale thermometry, it may also be possible to engineer biol. processes at the subcellular level. Here the authors demonstrate a new approach to nanoscale thermometry that uses coherent manipulation of the electronic spin assocd. with nitrogen-vacancy color centers in diamond. The authors' technique makes it possible to detect temp. variations as small as 1.8 mK (a sensitivity of 9 mK Hz-1/2) in an ultrapure bulk diamond sample. Using nitrogen-vacancy centers in diamond nanocrystals (nanodiamonds), the authors directly measure the local thermal environment on length scales as short as 200 nm. Finally, by introducing both nanodiamonds and gold nanoparticles into a single human embryonic fibroblast, the authors demonstrate temp.-gradient control and mapping at the subcellular level, enabling unique potential applications in life sciences.
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    Tian, Y.; Nusantara, A. C.; Hamoh, T.; Mzyk, A.; Tian, X.; Perona Martinez, F.; Li, R.; Permentier, H. P.; Schirhagl, R. Functionalized fluorescent nanodiamonds for simultaneous drug delivery and quantum sensing in HeLa cells. ACS Appl. Mater. Interfaces 2022, 14 (34), 39265– 39273,  DOI: 10.1021/acsami.2c11688
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    25
    Functionalized Fluorescent Nanodiamonds for Simultaneous Drug Delivery and Quantum Sensing in HeLa Cells
    Tian, Yuchen; Nusantara, Anggrek C.; Hamoh, Thamir; Mzyk, Aldona; Tian, Xiaobo; Perona Martinez, Felipe; Li, Runrun; Permentier, Hjalmar P.; Schirhagl, Romana
    ACS Applied Materials & Interfaces (2022), 14 (34), 39265-39273CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)
    Here, we present multifunctional fluorescent nanodiamonds (FNDs) for simultaneous drug delivery and free radical detection. For this purpose, we modified FNDs contg. nitrogen vacancy (NV) centers with a diazoxide deriv. We found that our particles enter cells more easily and are able to deliver this cancer drug into HeLa cells. The particles were characterized by IR spectroscopy, dynamic light scattering, and secondary electron microscopy. Compared to the free drug, we observe a sustained release over 72 h rather than 12 h for the free drug. Apart from releasing the drug, with these particles, we can measure the drug's effect on free radical generation directly. This has the advantage that the response is measured locally, where the drug is released. These FNDs change their optical properties based on their magnetic surrounding. More specifically, we make use of a technique called relaxometry to detect spin noise from the free radical at the nanoscale with subcellular resoln. We further compared the results from our new technique with a conventional fluorescence assay for the detection of reactive oxygen species. This provides a new method to investigate the relationship between drug release and the response by the cell via radical formation or inhibition.
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    Norouzi, N.; Nusantara, A. C.; Ong, Y.; Hamoh, T.; Nie, L.; Morita, A.; Zhang, Y.; Mzyk, A.; Schirhagl, R. Relaxometry for detecting free radical generation during Bacteria’s response to antibiotics. Carbon 2022, 199, 444– 452,  DOI: 10.1016/j.carbon.2022.08.025
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    26
    Relaxometry for detecting free radical generation during Bacteria's response to antibiotics
    Norouzi, Neda; Nusantara, Anggrek Citra; Ong, Yori; Hamoh, Thamir; Nie, Linyan; Morita, Aryan; Zhang, Yue; Mzyk, Aldona; Schirhagl, Romana
    Carbon (2022), 199 (), 444-452CODEN: CRBNAH; ISSN:0008-6223. (Elsevier Ltd.)
    Free radical generation plays a key role in killing bacteria by antibiotics. However, radicals are short-lived and reactive, and thus difficult to detect for the state of the art. Here we use a technique which allows optical nanoscale magnetic resonance imaging (MRI) to detect radical generation on the scale of single bacteria. We demonstrate that the radical generation in Staphylococcus aureus increases in the presence of UV irradn. as well as vancomycin and is dependent on the antibiotics dose. With a method based on ensembles of nitrogen vacancy (NV) centers in diamond, we were able to follow the radical formation near individual bacteria over the whole duration of the expt. to reveal the dynamics of radical generation. Using this new approach, we obsd. free radical concns. within nanoscale voxels around the diamond particles and detd. its exact timing depending on the antibiotic dose. Since changes in the response to antibiotics emerge in only a few bacteria of the entire population, such a single-cell approach can prove highly valuable for research into drug resistance.
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    Wu, K.; Nie, L.; Nusantara, A. C.; Woudstra, W.; Vedelaar, T.; Sigaeva, A.; Schirhagl, R. Diamond Relaxometry as a Tool to Investigate the Free Radical Dialogue between Macrophages and Bacteria. ACS Nano 2023, 17 (2), 1100– 1111,  DOI: 10.1021/acsnano.2c08190
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    27
    Diamond Relaxometry as a Tool to Investigate the Free Radical Dialogue between Macrophages and Bacteria
    Wu, Kaiqi; Nie, Linyan; Nusantara, Anggrek C.; Woudstra, Willem; Vedelaar, Thea; Sigaeva, Alina; Schirhagl, Romana
    ACS Nano (2023), 17 (2), 1100-1111CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
    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 obsd. 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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  28. 28
    Reyes-San-Martin, C.; Hamoh, T.; Zhang, Y.; Berendse, L.; Klijn, C.; Li, R.; Llumbet, A. E.; Sigaeva, A.; Kawałko, J.; Mzyk, A.; Schirhagl, R. Nanoscale mri for selective labeling and localized free radical measurements in the acrosomes of single sperm cells. ACS Nano 2022, 16 (7), 10701– 10710,  DOI: 10.1021/acsnano.2c02511
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    28
    Nanoscale MRI for Selective Labeling and Localized Free Radical Measurements in the Acrosomes of Single Sperm Cells
    Reyes-San-Martin, Claudia; Hamoh, Thamir; Zhang, Yue; Berendse, Lotte; Klijn, Carline; Li, Runrun; Llumbet, Arturo E.; Sigaeva, Alina; Kawalko, Jakub; Mzyk, Aldona; Schirhagl, Romana
    ACS Nano (2022), 16 (7), 10701-10710CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
    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.
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    Shenderova, O. A.; Shames, A. I.; Nunn, N. A.; Torelli, M. D.; Vlasov, I.; Zaitsev, A. Synthesis, properties, and applications of fluorescent diamond particles. J. Vac. Sci. Technol., B: Nanotechnol. Microelectron.: Mater., Process., Meas., Phenom. 2019, 37 (3), 030802  DOI: 10.1116/1.5089898
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    29
    Review Article: Synthesis, properties, and applications of fluorescent diamond particles
    Shenderova, Olga A.; Shames, Alexander I.; Nunn, Nicholas A.; Torelli, Marco D.; Vlasov, Igor; Zaitsev, Alexander
    Journal of Vacuum Science & Technology, B: Nanotechnology & Microelectronics: Materials, Processing, Measurement, & Phenomena (2019), 37 (3), 030802/1-030802/27CODEN: JVSTCN; ISSN:2166-2746. (American Institute of Physics)
    A review. Diamond particles contg. color centers-fluorescent crystallog. defects embedded within the diamond lattice-outperform other classes of fluorophores by providing a combination of unmatched photostability, intriguing coupled magneto-optical properties, intrinsic biocompatibility, and outstanding mech. and chem. robustness. This exceptional combination of properties positions fluorescent diamond particles as unique fluorophores with emerging applications in a variety of fields, including bioimaging, ultrasensitive metrol. at the nanoscale, fluorescent tags in industrial applications, and even potentially as magnetic resonance imaging contrast agents. However, prodn. of fluorescent nanodiamond (FND) is nontrivial, since it requires irradn. with high-energy particles to displace carbon atoms and create vacancies-a primary constituent in the majority color centers. In this review, centrally focused on material developments, major steps of FND prodn. are discussed with emphasis on current challenges in the field and possible solns. The authors demonstrate how the combination of fluorescent spectroscopy and ESR provides valuable insight into the types of radiation-induced defects formed and their evolution upon thermal annealing, thereby guiding FND performance optimization. A recent breakthrough process allowing for prodn. of fluorescent diamond particles with vibrant blue, green, and red fluorescence is also discussed. Finally, the authors conclude with demonstrations of a few FND applications in the life science arena and in industry. (c) 2019 American Institute of Physics.
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    Ong, S. Y.; Van Harmelen, R. J. J.; Norouzi, N.; Offens, F.; Venema, I. M.; Najafi, M. H.; Schirhagl, R. Interaction of nanodiamonds with bacteria. Nanoscale 2018, 10 (36), 17117– 17124,  DOI: 10.1039/C8NR05183F
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    Interaction of nanodiamonds with bacteria
    Ong, S. Y.; van Harmelen, R. J. J.; Norouzi, N.; Offens, F.; Venema, I. M.; Habibi Najafi, M. B.; Schirhagl, R.
    Nanoscale (2018), 10 (36), 17117-17124CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)
    Nanocarbons come in many forms and among their applications is the engineering of biocompatible and antibacterial materials. Studies have shown that diamond nanoparticles might have the interesting combination of both properties: they are highly biocompatible, while surprisingly reducing bacterial viability or growth at the same time. In this article, we consider for the first time the interaction of milled HPHT nanodiamonds with bacteria. These nanoparticles are capable of hosting nitrogen-vacancy (NV) centers, which provide stable fluorescence with potential use in sensing applications. An initial study was performed to assess the interaction of partially oxidized monocryst. nanodiamonds with Gram pos. S. aureus ATCC 12600 and Gram neg. E. coli ATCC 8739. It was shown that for S. aureus ATCC 12600, the presence of these nanodiamonds leads to a sharp redn. of colony forming ability under optimal conditions. A different effect was obsd. on Gram neg. E. coli ATCC 8739, where no significant adverse effects of ND presence was obsd. The mode of interaction was further studied by electron microscopy and confocal microscopy. The effects of NDs on S. aureus viability were found to depend on many factors, including the concn. and size of nanoparticles, the suspension medium and incubation time.
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    Preparation of biodegradable cyclosporine nanoparticles by high-pressure emulsification-solvent evaporation process
    Jaiswal, Jagdish; Gupta, Suresh Kumar; Kreuter, Jorg
    Journal of Controlled Release (2004), 96 (1), 169-178CODEN: JCREEC; ISSN:0168-3659. (Elsevier)
    The cyclic endecapeptide cyclosporine (CsA), a potent immunosuppressive drug, was incorporated into biodegradable poly (DL-lactide-co-glycolide) (dl-PLG) 50/50, 65/35 and PEG 5000-70/30 dL-PLG to improve the oral bioavailability and pharmacokinetics. Nanoparticles were prepd. by a high-pressure emulsification-solvent evapn. (HPESE) process. The CsA-loaded nanoparticles were evaluated for particle size, zeta potential, surface morphol. by SEM, thermal characterizations by differential scanning calorimetry (DSC), encapsulation efficiency (E.E.%) and in vitro release. The amt. of CsA loaded into the nanoparticles was detd. using high-performance liq. chromatog. (HPLC) at a detection wavelength of 210 nm. The mobile phase was acetonitrile-water (70:30% vol./vol.) and flow rate was set at 1.5 mL min-1. The photon correlation spectroscopy showed that the particles size were <250 nm and polydispersity index (PI) <0.14. The zeta potential was pos. for 200 mg and neg. for 400 mg of polymer compn., resp. The SEM micrographs revealed that the nanoparticles were spherical and smooth. The drug loading was between 82% and 92%. Differential scanning calorimetry (DSC) studies did not show the melting endotherm for CsA in the drug-loaded nanoparticles. In-vitro release in intestinal fluid pH 6.8 (USP XXIV) showed a cumulative percent release of 30-45% CsA in 8 h. The physicochem. properties showed that the dl-PLG and PEG-DLPLG nanoparticles could be an effective carrier for oral CsA delivery. The reported method is easy, reproducible and can be automated for batch scale prodn.
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    Nature Protocols (2010), 5 (9), 1540-1551CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)
    Precision-cut tissue slices (PCTS) are viable ex vivo explants of tissue with a reproducible, well defined thickness. They represent a mini-model of the organ under study and contain all cells of the tissue in their natural environment, leaving intercellular and cell-matrix interactions intact, and are therefore highly appropriate for studying multicellular processes. PCTS are mainly used to study the metab. and toxicity of xenobiotics, but they are suitable for many other purposes. Here we describe the protocols to prep. and incubate rat and human liver and intestinal slices. Slices are prepd. from fresh liver by making a cylindrical core using a drill with a hollow bit, from which slices are cut with a specially designed tissue slicer. Intestinal tissue is embedded in cylinders of agarose before slicing. Slices remain viable for 24 h (intestine) and up to 96 h (liver) when incubated in 6- or 12-well plates under 95% O2/5% CO2 atm.
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    Foroozandeh, P.; Aziz, A. A. Insight into cellular uptake and intracellular trafficking of nanoparticles. Nanoscale Res. Lett. 2018, 13, 339,  DOI: 10.1186/s11671-018-2728-6
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    Insight into Cellular Uptake and Intracellular Trafficking of Nanoparticles
    Foroozandeh Parisa; Aziz Azlan Abdul; Aziz Azlan Abdul
    Nanoscale research letters (2018), 13 (1), 339 ISSN:1931-7573.
    Nanoparticle science is rapidly changing the landscape of various scientific fields and defining new technological platforms. This is perhaps even more evident in the field of nanomedicine whereby nanoparticles have been used as a tool for the treatment and diagnosis of many diseases. However, despite the tremendous benefit conferred, common pitfalls of this technology is its potential short and long-term effects on the human body. To understand these issues, many scientific studies have been carried out. This review attempts to shed light on some of these studies and its outcomes. The topics that were examined in this review include the different possible uptake pathways of nanoparticles and intracellular trafficking routes. Additionally, the effect of physicochemical properties of nanoparticle such as size, shape, charge and surface chemistry in determining the mechanism of uptake and biological function of nanoparticles are also addressed.
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    van der Laan, K. J.; Naulleau, J.; Damle, V. G.; Sigaeva, A.; Jamot, N.; Perona-Martinez, F. P.; Chipaux, M.; Schirhagl, R. Toward using fluorescent nanodiamonds to study chronological aging in Saccharomyces cerevisiae. Anal. Chem. 2018, 90 (22), 13506– 13513,  DOI: 10.1021/acs.analchem.8b03431
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    Towards Using Fluorescent Nanodiamonds To Study Chronological Aging in Saccharomyces cerevisiae
    van der Laan, Kiran J.; Naulleau, Julie; Damle, Viraj G.; Sigaeva, Alina; Jamot, Nicolas; Perona-Martinez, Felipe P.; Chipaux, Mayeul; Schirhagl, Romana
    Analytical Chemistry (Washington, DC, United States) (2018), 90 (22), 13506-13513CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    One of the theories aiming to explain cellular aging is the free radical theory of aging, which describes the possible role of increased prodn. 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 mol. 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 chronol. 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.
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    Vaijayanthimala, V.; Tzeng, Y. K.; Chang, H. C.; Li, C. L. The biocompatibility of fluorescent nanodiamonds and their mechanism of cellular uptake. Nanotechnology 2009, 20 (42), 425103  DOI: 10.1088/0957-4484/20/42/425103
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    The biocompatibility of fluorescent nanodiamonds and their mechanism of cellular uptake
    Vaijayanthimala, Vairakkannu; Tzeng, Yan-Kai; Chang, Huan-Cheng; Li, Chung-Leung
    Nanotechnology (2009), 20 (42), 425103/1-425103/9CODEN: NNOTER; ISSN:1361-6528. (Institute of Physics Publishing)
    The labeling of cells with fluorescent nanoparticles is promising for various biomedical applications. The objective of this study is to evaluate the biocompatibility and the mechanism of the cellular uptake of fluorescent nanodiamonds (FNDs) in cancer cells (HeLa) and pre-adipocytes (3T3-L1). With flow cytometry and the use of a battery of metabolic and cytoskeletal inhibitors, we found that the mechanism of the FND uptake in both cells is by energy-dependent clathrin-mediated endocytosis. In addn., the surface charge of FND influences its cellular uptake, as the uptake of poly-L-lysine-coated FNDs is better than that of oxidative-acid-purified FNDs at the same concn. in regular medium with or without serum. We also confirm that the proliferative potential of FND-treated and untreated cells does not exhibit any significant differences when measured at bulk cultures, and more stringently at clonal cell d. Further biocompatibility studies indicate that the in vitro differentiation of 3T3-L1 pre-adipocytes and 489-2 osteoprogenitors is not affected by the FND treatment. Our results show that FNDs are biocompatible and ideal candidates for potential applications in human stem cell research.
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    Hemelaar, S. R.; Saspaanithy, B.; L’hommelet, S. R.; Perona Martinez, F. P.; Van der Laan, K. J.; Schirhagl, R. The response of HeLa cells to fluorescent nanodiamond uptake. Sensors 2018, 18 (2), 355,  DOI: 10.3390/s18020355
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    The response of hela cells to fluorescent nanodiamond uptake
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    Sensors (2018), 18 (2), 355/1-355/15CODEN: SENSC9; ISSN:1424-8220. (MDPI AG)
    Fluorescent nanodiamonds are promising probes for nanoscale magnetic resonance measurements. Their phys. properties predict them to have particularly useful applications in intracellular anal. Before using them in intracellular expts. however, it should be clear whether diamond particles influence cell biol. While cytotoxicity has already been ruled out in previous studies, we consider the non-fatal influence of fluorescent nanodiamonds on the formation of reactive oxygen species (an important stress indicator and potential target for intracellular sensing) for the first time. We investigated the influence of different sizes, shapes and concns. of nanodiamonds on the genetic and protein level involved in oxidative stress-related pathways of the HeLa cell, an important model cell line in research. The changes in viability of the cells and the difference in intracellular levels of free radicals, after diamond uptake, are surprisingly small. At lower diamond concns., the cellular metab. cannot be distinguished from that of untreated cells. This research supports the claims of non-toxicity and includes less obvious non-fatal responses. Finally, we give a handhold concerning the diamond concn. and size to use for non-toxic, intracellular measurements in favor of (cancer) research in HeLa cells.
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    Zipfel, W. R.; Williams, R. M.; Christie, R.; Nikitin, A. Y.; Hyman, B. T.; Webb, W. W. Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation. Proc. Natl. Acad. Sci. U.S.A. 2003, 100 (12), 7075– 7080,  DOI: 10.1073/pnas.0832308100
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    Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation
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    Multicolor nonlinear microscopy of living tissue using two- and three-photon-excited intrinsic fluorescence combined with second harmonic generation by supermol. structures produces images with the resoln. and detail of std. histol. without the use of exogenous stains. Imaging of intrinsic indicators within tissue, such as NAD, retinol, indoleamines, and collagen provides crucial information for physiol. and pathol. The efficient application of multiphoton microscopy to intrinsic imaging requires knowledge of the nonlinear optical properties of specific cell and tissue components. Here we compile and demonstrate applications involving a range of intrinsic mols. and mol. assemblies that enable direct visualization of tissue morphol., cell metab., and disease states such as Alzheimer's disease and cancer.
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    Jun, Y. W.; Kim, H. R.; Reo, Y. J.; Dai, M.; Ahn, K. H. Addressing the autofluorescence issue in deep tissue imaging by two-photon microscopy: the significance of far-red emitting dyes. Chem. Sci. 2017, 8 (11), 7696– 7704,  DOI: 10.1039/C7SC03362A
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    Addressing the autofluorescence issue in deep tissue imaging by two-photon microscopy: the significance of far-red emitting dyes
    Jun, Yong Woong; Kim, Hye Rim; Reo, Ye Jin; Dai, Mingchong; Ahn, Kyo Han
    Chemical Science (2017), 8 (11), 7696-7704CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
    The fluorescence imaging of tissue is essential for studying biol. events beyond the cellular level. Two-photon microscopy based on the nonlinear light absorption of fluorescent dyes is a viable tool for the high resoln. imaging of tissue. A key limitation for deep tissue imaging is the autofluorescence from intrinsic biomols. Here, we report a systematic study that discloses relative autofluorescence interference, which is dependent on the type of tissue and the excitation and emission wavelengths in two-photon imaging. Among the brain, kidney, liver, lung, and spleen mouse tissues examd., the kidney tissue exhibited prominent autofluorescence followed by the liver and others. Notably, regardless of the tissue type, prominent autofluorescence is obsd. not only from the green emission channel but also from the yellow emission channel where common two-photon absorbing dyes also emit, whereas there is minimal autofluorescence from the red channel. The autofluorescence is slightly influenced by the excitation wavelength. Toward minimal autofluorescence, we developed a new class of two-photon absorbing dyes that are far-red emitting, water-sol., and very bright inside cells as well as in tissue. A comparative assessment of the imaging depth, which is dependent on the three selected dyes that emit in the blue-green, yellow, and far-red regions, shows the importance of far-red emitting dyes for deep tissue imaging.
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    Li, R.; Vedelaar, T.; Mzyk, A.; Morita, A.; Padamati, S. K.; Schirhagl, R. Following Polymer Degradation with Nanodiamond Magnetometry. ACS Sens. 2022, 7 (1), 123– 130,  DOI: 10.1021/acssensors.1c01782
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    Following Polymer Degradation with Nanodiamond Magnetometry
    Li, Runrun; Vedelaar, Thea; Mzyk, Aldona; Morita, Aryan; Padamati, Sandeep Kumar; Schirhagl, Romana
    ACS Sensors (2022), 7 (1), 123-130CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)
    Degradable polymers are widely used in the biomedical fields due to non-toxicity and great biocompatibility and biodegradability, and it is crucial to understand how they degrade. These polymers are exposed to various biochem. media in medical practice. Hence, it is important to precisely follow the degrdn. of the polymer in real time. In this study, we made use of diamond magnetometry for the first time to track polymer degrdn. with nanoscale precision. The method is based on a fluorescent defect in nanodiamonds, which changes its optical properties based on its magnetic surrounding. Since optical signals can be read out more sensitively than magnetic signals, this method allows unprecedented sensitivity. We used a specific mode of diamond magnetometry called relaxometry or T1 measurements. These are sensitive to magnetic noise and thus can detect paramagnetic species (gadolinium in this case). Nanodiamonds were incorporated into polylactic acid (PLA) films and PLA nanoparticles in order to follow polymer degrdn. However, in principle, they can be incorporated into other polymers too. We found that T1 consts. decreased gradually with the erosion of the film exposed to an alk. condition. In addn., the mobility of nanodiamonds increased, which allows us to est. polymer viscosity. The degrdn. rates obtained using this approach were in good agreement with data obtained by quartz crystal microbalance, Fourier-transform IR spectroscopy, and at. force microscopy.
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    Prabhakar, N.; Khan, M. H.; Peurla, M.; Chang, H. C.; Hänninen, P. E.; Rosenholm, J. M. Intracellular trafficking of fluorescent nanodiamonds and regulation of their cellular toxicity. ACS Omega 2017, 2 (6), 2689– 2693,  DOI: 10.1021/acsomega.7b00339
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    Intracellular Trafficking of Fluorescent Nanodiamonds and Regulation of Their Cellular Toxicity
    Prabhakar, Neeraj; Khan, Meraj H.; Peurla, Markus; Chang, Huan-Cheng; Hanninen, Pekka E.; Rosenholm, Jessica M.
    ACS Omega (2017), 2 (6), 2689-2693CODEN: ACSODF; ISSN:2470-1343. (American Chemical Society)
    Cellular management of fluorescent nanodiamonds (FNDs) has been studied for better understanding in designing potential applications of FNDs in biomedicine. The FNDs have been shown to be photostable probes for bioimaging and thus are well suited for e.g. long term tracking purposes. FNDs also exhibit good biocompatibility and in general, low toxicity for cell labeling. To demonstrate the underlying mechanism of cells coping the low but potentially toxic effects by non-degradable FNDs, the authors have studied their temporal intracellular trafficking. FNDs are localized as distinct populations inside cells in early endosomes, lysosomes, and in proximity to the plasma membrane. The localization of FNDs in early endosomes suggests the internalization of FNDs, and lysosomal localization in turn can be interpreted as a pre-state for exocytosis via lysosomal degrdn. pathway. The endocytosis and exocytosis appears to be occurring simultaneously in the authors' observations. The mechanism of continuous endocytosis and exocytosis of FNDs could be necessary for cells to maintain normal proliferation. Further, a 120 h cell growth assay was performed to verify the long-term biocompatibility of FNDs for cellular studies.
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    Nie, L.; Zhang, Y.; Li, L.; van Rijn, P.; Schirhagl, R. pH sensitive dextran coated fluorescent nanodiamonds as a biomarker for hela cells endocytic pathway and increased cellular uptake. Nanomaterials 2021, 11 (7), 1837,  DOI: 10.3390/nano11071837
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    The pH Sensitive Dextran Coated Fluorescent Nanodiamonds as a Biomarker for HeLa Cells Endocytic Pathway and Increased Cellular Uptake
    Nie, Linyan; Zhang, Yue; Li, Lei; van Rijn, Patrick; Schirhagl, Romana
    Nanomaterials (2021), 11 (7), 1837CODEN: NANOKO; ISSN:2079-4991. (MDPI AG)
    Fluorescent nanodiamonds are a useful for biosensing of intracellular signaling networks or environmental changes (such as temp., pH or free radical generation). HeLa cells are interesting to study with these nanodiamonds since they are a model cell system that is widely used to study cancer-related diseases. However, they only internalize low nos. of nanodiamond particles very slowly via the endocytosis pathway. In this work, we show that pH-sensitive, dextran-coated fluorescent nanodiamonds can be used to visualize this pathway. Addnl., this coating improved diamond uptake in HeLa cells by 5.3 times (*** p < 0.0001) and decreased the required time for uptake to only 30 min. We demonstrated further that nanodiamonds enter HeLa cells via endolysosomes and are eventually expelled by cells.
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    Sigaeva, A.; Hochstetter, A.; Bouyim, S.; Chipaux, M.; Stejfova, M.; Cigler, P.; Schirhagl, R. Single-Particle Tracking and Trajectory Analysis of Fluorescent Nanodiamonds in Cell-Free Environment and Live Cells. Small 2022, 18 (39), 2201395  DOI: 10.1002/smll.202201395
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    Single-Particle Tracking and Trajectory Analysis of Fluorescent Nanodiamonds in Cell-Free Environment and Live Cells
    Sigaeva, Alina; Hochstetter, Axel; Bouyim, Sighom; Chipaux, Mayeul; Stejfova, Miroslava; Cigler, Petr; Schirhagl, Romana
    Small (2022), 18 (39), 2201395CODEN: SMALBC; ISSN:1613-6810. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Diamond magnetometry can provide new insights on the prodn. of free radicals inside live cells due to its high sensitivity and spatial resoln. However, the measurements often lack intracellular context for the recorded signal. In this paper, the possible use of single-particle tracking and trajectory anal. of fluorescent nanodiamonds (FNDs) to bridge that gap is explored. It starts with simulating a set of different possible scenarios of a particles movement, reflecting different modes of motion, degrees of confinement, as well as shapes and sizes of that confinement. Then, the insights from the anal. of the simulated trajectories are applied to describe the movement of FNDs in glycerol solns. It is shown that the measurements are in good agreement with the previously reported findings and that trajectory anal. 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.
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Cite this: Anal. Chem. 2023, 95, 35, 13046–13054
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  • Abstract

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    Figure 1

    Figure 1. Preparation of PLA-FND particles, in vitro evaluation, and cellular tracking. (a) PLA and FNDs in chloroform are added to PVA in aqueous solution. (b) Tip sonication facilitates the formation of an emulsion, and the presence of PVA stabilizes the emulsion and leads to a uniform particle size distribution. (c) Particles were collected by centrifugation after magnetic stirring overnight. (d) Particle internalization and location within the cell and quantitative analysis. (e) Particle internalization in mouse liver slice. (f) Diamonds within polymer particles were tracked using a custom-built confocal microscope.

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    Figure 2

    Figure 2. FNDs, PLA-FNDs, and PLA nanoparticles were taken up by macrophages after 2 h of incubation with particles plus 0, 6, and 24 h of incubation in the medium without particles. Red represent the FNDs, green represents the cytoskeleton, and blue represents the nucleus. Merged images are shown. The red fluorescence of FNDs and PLA-FNDs was seen after nanoparticles were taken up. In the PLA samples, we did not observe any red fluorescence inside the cells, indicating that PLA itself has no autofluorescence across the visible wavelength we used.

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    Figure 3

    Figure 3. Number of particles in each cell decreases over time; however, noticeable aggregation occurred. All data were analyzed from at least 50 cells. (A) The spatial distribution of particles per cell is shown in violin plots. Wider sections mean a higher probability that the number of particles per cell is distributed there. Black dotted lines from top to bottom mean third quartile, median, and first quartile, respectively. (B) The particle volume is shown in box plots. The box covers the 5–95 percentile of the data, and the horizontal lines that split the box are the median. (C) Cell numbers at varying incubation times are shown in scatter plots. Black lines are mean ± standard deviation.

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    Figure 4

    Figure 4. PLA-FNDs (A) and FNDs (B) were also observed in mouse liver tissue after 48 h of incubation (red: FNDs, green: tissue autofluorescence).

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    Figure 5

    Figure 5. Colocalization of PLA and PLA-FNDs with early endosome compartments labeled by EEA1 antibody after 0, 6, and 24 h of incubation (red: FNDs, green: early endosomes, blue: nucleus). Merged images are shown. All data were analyzed from at least 50 cells.

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    Figure 6

    Figure 6. Colocalization of FNDs and PLA-FNDs with lysosome compartments labeled by LAMP-1 antibodies after 0, 6, and 24 h of incubation (red: FNDs, green: late endosomes and lysosomes, blue: nucleus). Merged images are shown. All data were obtained from at least 50 cells.

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    Figure 7

    Figure 7. Quantitative colocalization analysis of ingested FNDs and PLA-FNDs with endosomes and lysosomes from confocal microscopy images. All data are shown as mean ± standard deviation for a total of 50 cells at least. (A) Quantitative analysis of FNDs, PLA-FND colocalization with early endosomes labeled with EEA1. The results indicate no statistically significant difference between the different groups. (B) Quantitative analysis of FNDs, PLA-FND colocalization with late endosomes and lysosomes labeled with LAMP-1. The results indicated statistically significant differences between FNDs or PLA-FNDs.

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    Figure 8

    Figure 8. Mean diffusion coefficient during 8 h measurements in the xy-plane. Three FNDs and three PLA-FNDs were tested. Black dotted lines indicate the median diffusion coefficient of FBS-coated 70 and 120 nm FNDs in HeLa cells from previous work (43) (different bare particles are numbered as FND 01, FND 02, and FND 03, while PLA-FND 01, PLA-FND 02, and PLA-FND 03 indicate FNDs that are in PLA particles).

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      Micro- and nanoplastics - current state of knowledge with the focus on oral uptake and toxicity
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      Nanoscale Advances (2020), 2 (10), 4350-4367CODEN: NAADAI; ISSN:2516-0230. (Royal Society of Chemistry)
      A review. The prodn. and use of plastics has constantly increased over the last 30 years. Over one third of the plastics is used in disposables, which are discarded within three years of their prodn. Despite efforts towards recycling, a substantial vol. of debris has accumulated in the environment and is slowly degraded to micro- and nanoplastics by weathering and aging. It has recently been discovered that these small particles can enter the food chain, as for example demonstrated by the detection of microplastic particles in honey, beer, salt, sea food and recently in mineral water. Human exposure has further been documented by the detection of plastic microparticles in human feces. Potential toxic consequences of oral exposure to small plastic particles are discussed. Due to lacking data concerning exposure, biodistribution and related effects, the risk assessment of micro- and nanoplastics is still not possible. This review focuses on the oral uptake of plastic and polymer micro- and nanoparticles. Oral exposure, particle fate, changes of particle properties during ingestion and gastrointestinal digestion, and uptake and transport at the intestinal epithelium are reviewed in detail. Moreover, the interaction with intestinal and liver cells and possibly resulting toxicity are highlighted.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhslajurzK&md5=686225513932d3499a529117f53a9f54
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      Mariano, S.; Tacconi, S.; Fidaleo, M.; Rossi, M.; Dini, L. Micro and nanoplastics identification: classic methods and innovative detection techniques. Front. Toxicol. 2021, 3, 636640  DOI: 10.3389/ftox.2021.636640
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      Micro and Nanoplastics Identification: Classic Methods and Innovative Detection Techniques
      Mariano Stefania; Tacconi Stefano; Fidaleo Marco; Dini Luciana; Rossi Marco; Rossi Marco; Dini Luciana; Rossi Marco; Dini Luciana
      Frontiers in toxicology (2021), 3 (), 636640 ISSN:.
      Micro and nanoplastics are fragments with dimensions less than a millimeter invading all terrestrial and marine environments. They have become a major global environmental issue in recent decades and, indeed, recent scientific studies have highlighted the presence of these fragments all over the world even in environments that were thought to be unspoiled. Analysis of micro/nanoplastics in isolated samples from abiotic and biotic environmental matrices has become increasingly common. Hence, the need to find valid techniques to identify these micro and nano-sized particles. In this review, we discuss the current and potential identification methods used in microplastic analyses along with their advantages and limitations. We discuss the most suitable techniques currently available, from physical to chemical ones, as well as the challenges to enhance the existing methods and develop new ones. Microscopical techniques (i.e., dissect, polarized, fluorescence, scanning electron, and atomic force microscopy) are one of the most used identification methods for micro/nanoplastics, but they have the limitation to produce incomplete results in analyses of small particles. At present, the combination with chemical analysis (i.e., spectroscopy) overcome this limit together with recently introduced alternative approaches. For example, holographic imaging in microscope configuration images microplastics directly in unfiltered water, thus discriminating microplastics from diatoms and differentiates different sizes, shapes, and plastic types. The development of new analytical instruments coupled with each other or with conventional and innovative microscopy could solve the current problems in the identification of micro/nanoplastics.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB2Mzks1GrsQ%253D%253D&md5=d9648c723223e35f4ac62484af37eae8
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      Single-particle analysis of micro/nanoplastics by SEM-Raman technique
      Li, Gang; Yang, Zhiruo; Pei, Zhiguo; Li, Yingming; Yang, Ruiqiang; Liang, Yong; Zhang, Qinghua; Jiang, Guibin
      Talanta (2022), 249 (), 123701CODEN: TLNTA2; ISSN:0039-9140. (Elsevier B.V.)
      Micro/nanoplastics (MNPs) have received global concern due to their widespread contamination, ingestion in organisms, and the ability to cross the biol. barrier. Although MNPs have been detected in a variety of ecosystems, the identification of single MNPs remains an unsolved challenge. Herein, for the first time, scanning electron microscope (SEM) coupled with surface-enhanced Raman spectroscopy (SERS), which combined the advantages of ultrahigh spatial resoln. of SEM and structural fingerprint of Raman spectroscopy, was proposed to identify MNPs at single-particle level. Under the optimum conditions, the polystyrene (PS) MNPs with sizes of 500 nm and 1μm were identified by the image of SEM and fingerprint peaks of Raman spectroscopy. Addnl., the applicability of the method in different sample matrixes and for other types of MNPs such as poly-Me methacrylate (PMMA) with the sizes of 300 nm, 1μm were validated. This method is simple, rapid and effective and is likely to provide an essential tool to identify other micro/nanoparticles in addn. to MNPs.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xhs1Knu73E&md5=32b019127ba2566688c0251da84aa2a0
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      Identification and quantification of microplastics using Nile Red staining
      Shim, Won Joon; Song, Young Kyoung; Hong, Sang Hee; Jang, Mi
      Marine Pollution Bulletin (2016), 113 (1-2), 469-476CODEN: MPNBAZ; ISSN:0025-326X. (Elsevier Ltd.)
      We investigated the applicability of Nile Red (NR), a fluorescent dye, for microplastic anal., and detd. the optimal staining conditions. Five mg/L NR soln. in n-hexane effectively stained plastics, and they were easily recognized in green fluorescence. The NR staining method was successfully applied to micro-sized polyethylene, polypropylene, polystyrene, polycarbonate, polyurethane, and poly(ethylene-vinyl acetate), except for polyvinylchloride, polyamide and polyester. The recovery rate of polyethylene (100-300 μm) spiked to pretreated natural sand was 98% in the NR stating method, which was not significantly (p < 0.05) different with FT-IR identification. The NR staining method was suitable for discriminating fragmented polypropylene particles from large nos. of sand particles in lab. weathering test samples. The method is straightforward and quick for identifying and quantifying polymer particles in the lab. controlled samples. Further studies, however, are necessary to investigate the application of NR staining to field samples with org. remnants.
      >> More from SciFinder ®
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      9
      In situ surface-enhanced Raman spectroscopy for detecting microplastics and nanoplastics in aquatic environments
      Lv, Lulu; He, Lei; Jiang, Shiqi; Chen, Jinjun; Zhou, Chunxia; Qu, Junhao; Lu, Yuqin; Hong, Pengzhi; Sun, Shengli; Li, Chengyong
      Science of the Total Environment (2020), 728 (), 138449CODEN: STENDL; ISSN:0048-9697. (Elsevier B.V.)
      Micro- and nano-plastics detection in environmental matrixes, particularly in-situ plastic particles in aquatic environments, still is challenged due to limitations of existing methods, instruments, and plastic particle sizes. This work evaluated the potential of surface-enhanced Raman spectroscopy to analyze micro- and nano-plastics. Conditions for different tests (sample:Ag colloid vol. ratio, NaCl and sample concns.) were studied to assess micro- and nano-plastics (polystyrene [PS], polyethylene [PE], polypropylene [PP]) in pure water and seawater. A SERS-based method which uses Ag colloid as the active substrate was developed to qual. analyze micro- and nano-plastics in aquatic environments. Micro- and nano-plastic particle sizes were 100 and 500 nm and 10μm. Micro- and nano-plastics Raman signals in pure water and seawater both showed good enhancement efficiency. The optimal enhancement factor was 4 x 104. The SERS-based detection method overcame limitations of micro- and nano-plastics in liqs. and detects 100 nm plastics down to 40μg/mL. It provides greater possibilities for future rapid micro- and nano-plastics detection in aquatic environments.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXotVCqsb4%253D&md5=d6b2934a206f9267a3298f997e969fa4
    10. 10
      Vaijayanthimala, V.; Cheng, P. Y.; Yeh, S. H.; Liu, K. K.; Hsiao, C. H.; Chao, J. I.; Chang, H. C. The long-term stability and biocompatibility of fluorescent nanodiamond as an in vivo contrast agent. Biomaterials 2012, 33 (31), 7794– 7802,  DOI: 10.1016/j.biomaterials.2012.06.084
      10
      The long-term stability and biocompatibility of fluorescent nanodiamond as an in vivo contrast agent
      Vaijayanthimala, V.; Cheng, Po-Yun; Yeh, Shih-Hua; Liu, Kuang-Kai; Hsiao, Cheng-Hsiang; Chao, Jui-I.; Chang, Huan-Cheng
      Biomaterials (2012), 33 (31), 7794-7802CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)
      Nanocarbon is a promising type of biomaterial for diagnostic and therapeutic applications. Fluorescent nanodiamond (FND) contg. nitrogen-vacancy centers as built-in fluorophores is a new addn. to the nanocarbon family. Here, we study the long-term stability and biocompatibility of 100-nm FNDs in rats through i.p. injection over 5 mo and develop the potential application of this biomaterial for sentinel lymph node mapping in a mouse model. From both in vivo and ex vivo fluorescence imaging as well as transmission electron microscopy, we found that the intradermally administered FND particles can be drained from the injection sites by macrophages and selectively accumulated in the axillary lymph nodes of the treated mice. Our measurements of water consumption, fodder consumption, body wt., and organ index showed no significant difference between control and FND-treated groups of the rats. Histopathol. anal. of various tissues and organs indicated that FNDs are non-toxic even when a large quantity, up to 75 mg/kg body wt., of the particles was administered i.p. to the living animals. With the properties of wide-ranging biocompatibility and perfect chem. and photophys. stability, FND is well suited for use as a contrast agent for long-term in vivo imaging.
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    11. 11
      Vaijayanthimala, V.; Tzeng, Y. K.; Chang, H. C.; Li, C. L. The biocompatibility of fluorescent nanodiamonds and their mechanism of cellular uptake. Nanotechnology 2009, 20 (42), 425103  DOI: 10.1088/0957-4484/20/42/425103
      11
      The biocompatibility of fluorescent nanodiamonds and their mechanism of cellular uptake
      Vaijayanthimala, Vairakkannu; Tzeng, Yan-Kai; Chang, Huan-Cheng; Li, Chung-Leung
      Nanotechnology (2009), 20 (42), 425103/1-425103/9CODEN: NNOTER; ISSN:1361-6528. (Institute of Physics Publishing)
      The labeling of cells with fluorescent nanoparticles is promising for various biomedical applications. The objective of this study is to evaluate the biocompatibility and the mechanism of the cellular uptake of fluorescent nanodiamonds (FNDs) in cancer cells (HeLa) and pre-adipocytes (3T3-L1). With flow cytometry and the use of a battery of metabolic and cytoskeletal inhibitors, we found that the mechanism of the FND uptake in both cells is by energy-dependent clathrin-mediated endocytosis. In addn., the surface charge of FND influences its cellular uptake, as the uptake of poly-L-lysine-coated FNDs is better than that of oxidative-acid-purified FNDs at the same concn. in regular medium with or without serum. We also confirm that the proliferative potential of FND-treated and untreated cells does not exhibit any significant differences when measured at bulk cultures, and more stringently at clonal cell d. Further biocompatibility studies indicate that the in vitro differentiation of 3T3-L1 pre-adipocytes and 489-2 osteoprogenitors is not affected by the FND treatment. Our results show that FNDs are biocompatible and ideal candidates for potential applications in human stem cell research.
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    12. 12
      Hsu, T. C.; Liu, K. K.; Chang, H. C.; Hwang, E.; Chao, J. I. Labeling of neuronal differentiation and neuron cells with biocompatible fluorescent nanodiamonds. Sci. Rep. 2014, 4 (1), 5004  DOI: 10.1038/srep05004
      12
      Labeling of neuronal differentiation and neuron cells with biocompatible fluorescent nanodiamonds
      Hsu, Tzu-Chia; Liu, Kuang-Kai; Chang, Huan-Cheng; Hwang, Eric; Chao, Jui-I.
      Scientific Reports (2014), 4 (), 5004CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
      Nanodiamond is a promising carbon nanomaterial developed for biomedical applications. Here, we show fluorescent nanodiamond (FND) with the biocompatible properties that can be used for the labeling and tracking of neuronal differentiation and neuron cells derived from embryonal carcinoma stem (ECS) cells. The fluorescence intensities of FNDs were increased by treatment with FNDs in both the mouse P19 and human NT2/D1 ECS cells. FNDs were taken into ECS cells; however, FNDs did not alter the cellular morphol. and growth ability. Moreover, FNDs did not change the protein expression of stem cell marker SSEA-1 of ECS cells. The neuronal differentiation of ECS cells could be induced by retinoic acid (RA). Interestingly, FNDs did not affect on the morphol. alteration, cytotoxicity and apoptosis during the neuronal differentiation. Besides, FNDs did not alter the cell viability and the expression of neuron-specific marker β-III-tubulin in these differentiated neuron cells. The existence of FNDs in the neuron cells can be identified by confocal microscopy and flow cytometry. Together, FND is a biocompatible and readily detectable nanomaterial for the labeling and tracking of neuronal differentiation process and neuron cells from stem cells.
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    13. 13
      van der Laan, K.; Hasani, M.; Zheng, T.; Schirhagl, R. Nanodiamonds for in vivo applications. Small 2018, 14 (19), 1703838  DOI: 10.1002/smll.201703838
      There is no corresponding record for this reference.
    14. 14
      Haziza, S.; Mohan, N.; Loe-Mie, Y.; Lepagnol-Bestel, A. M.; Massou, S.; Adam, M. P.; Le, X. L.; Viard, J.; Plancon, C.; Daudin, R.; Koebel, P. Fluorescent nanodiamond tracking reveals intraneuronal transport abnormalities induced by brain-disease-related genetic risk factors. Nat. Nanotechnol. 2017, 12 (4), 322– 328,  DOI: 10.1038/nnano.2016.260
      14
      Fluorescent nanodiamond tracking reveals intraneuronal transport abnormalities induced by brain-disease-related genetic risk factors
      Haziza, Simon; Mohan, Nitin; Loe-Mie, Yann; Lepagnol-Bestel, Aude-Marie; Massou, Sophie; Adam, Marie-Pierre; Le, Xuan Loc; Viard, Julia; Plancon, Christine; Daudin, Rachel; Koebel, Pascale; Dorard, Emilie; Rose, Christiane; Hsieh, Feng-Jen; Wu, Chih-Che; Potier, Brigitte; Herault, Yann; Sala, Carlo; Corvin, Aiden; Allinquant, Bernadette; Chang, Huan-Cheng; Treussart, Francois; Simonneau, Michel
      Nature Nanotechnology (2017), 12 (4), 322-328CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
      Brain diseases such as autism and Alzheimer's disease (each inflicting >1% of the world population) involve a large network of genes displaying subtle changes in their expression. Abnormalities in intraneuronal transport have been linked to genetic risk factors found in patients, suggesting the relevance of measuring this key biol. process. However, current techniques are not sensitive enough to detect minor abnormalities. Here the authors report a sensitive method to measure the changes in intraneuronal transport induced by brain-disease-related genetic risk factors using fluorescent nanodiamonds (FNDs). The authors show that the high brightness, photostability and absence of cytotoxicity allow FNDs to be tracked inside the branches of dissocd. neurons with a spatial resoln. of 12 nm and a temporal resoln. of 50 ms. As proof of principle, the authors applied the FND tracking assay on two transgenic mouse lines that mimic the slight changes in protein concn. (∼30%) found in the brains of patients. In both cases, the authors show that the FND assay is sufficiently sensitive to detect these changes.
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    15. 15
      Wu, T. J.; Tzeng, Y. K.; Chang, W. W.; Cheng, C. A.; Kuo, Y.; Chien, C. H.; Chang, H. C.; Yu, J. Tracking the engraftment and regenerative capabilities of transplanted lung stem cells using fluorescent nanodiamonds. Nat. Nanotechnol. 2013, 8 (9), 682– 689,  DOI: 10.1038/nnano.2013.147
      15
      Tracking the engraftment and regenerative capabilities of transplanted lung stem cells using fluorescent nanodiamonds
      Wu, Tsai-Jung; Tzeng, Yan-Kai; Chang, Wei-Wei; Cheng, Chi-An; Kuo, Yung; Chien, Chin-Hsiang; Chang, Huan-Cheng; Yu, John
      Nature Nanotechnology (2013), 8 (9), 682-689CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
      Lung stem/progenitor cells are potentially useful for regenerative therapy, for example in repairing damaged or lost lung tissue in patients. Several optical imaging methods and probes have been used to track how stem cells incorporate and regenerate themselves in vivo over time. However, these approaches are limited by photobleaching, toxicity and interference from background tissue autofluorescence. Here we show that fluorescent nanodiamonds, in combination with fluorescence-activated cell sorting, fluorescence lifetime imaging microscopy and immunostaining, can identify transplanted CD45-CD54+CD157+ lung stem/progenitor cells in vivo, and track their engraftment and regenerative capabilities with single-cell resoln. Fluorescent nanodiamond labeling did not eliminate the cells' properties of self-renewal and differentiation into type I and type II pneumocytes. Time-gated fluorescence imaging of tissue sections of naphthalene-injured mice indicates that the fluorescent nanodiamond-labeled lung stem/progenitor cells preferentially reside at terminal bronchioles of the lungs for 7 days after i.v. transplantation.
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    16. 16
      Sigaeva, A.; Hochstetter, A.; Bouyim, S.; Chipaux, M.; Stejfova, M.; Cigler, P.; Schirhagl, R. Single-Particle Tracking and Trajectory Analysis of Fluorescent Nanodiamonds in Cell-Free Environment and Live Cells. Small 2022, 18 (39), 2201395  DOI: 10.1002/smll.202201395
      16
      Single-Particle Tracking and Trajectory Analysis of Fluorescent Nanodiamonds in Cell-Free Environment and Live Cells
      Sigaeva, Alina; Hochstetter, Axel; Bouyim, Sighom; Chipaux, Mayeul; Stejfova, Miroslava; Cigler, Petr; Schirhagl, Romana
      Small (2022), 18 (39), 2201395CODEN: SMALBC; ISSN:1613-6810. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Diamond magnetometry can provide new insights on the prodn. of free radicals inside live cells due to its high sensitivity and spatial resoln. However, the measurements often lack intracellular context for the recorded signal. In this paper, the possible use of single-particle tracking and trajectory anal. of fluorescent nanodiamonds (FNDs) to bridge that gap is explored. It starts with simulating a set of different possible scenarios of a particles movement, reflecting different modes of motion, degrees of confinement, as well as shapes and sizes of that confinement. Then, the insights from the anal. of the simulated trajectories are applied to describe the movement of FNDs in glycerol solns. It is shown that the measurements are in good agreement with the previously reported findings and that trajectory anal. 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.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XitlWjurvK&md5=f7ff69e5276b7dae0d09d1e124a58ec7
    17. 17
      Hui, Y. Y.; Hsiao, W. W. W.; Haziza, S.; Simonneau, M.; Treussart, F.; Chang, H. C. Single particle tracking of fluorescent nanodiamonds in cells and organisms. Curr. Opin. Solid State Mater. Sci. 2017, 21 (1), 35– 42,  DOI: 10.1016/j.cossms.2016.04.002
      17
      Single particle tracking of fluorescent nanodiamonds in cells and organisms
      Hui, Yuen Yung; Hsiao, Wesley Wei-Wen; Haziza, Simon; Simonneau, Michel; Treussart, Francois; Chang, Huan-Cheng
      Current Opinion in Solid State & Materials Science (2017), 21 (1), 35-42CODEN: COSSFX; ISSN:1359-0286. (Elsevier Ltd.)
      Ever since the discovery of fullerenes in 1985, nanocarbon has demonstrated a wide range of applications in various areas of science and engineering. Compared with metal, oxide, and semiconductor nanoparticles, the carbon-based nanomaterials have distinct advantages in both biotechnol. and biomedical applications due to their inherent biocompatibility. Fluorescent nanodiamond (FND) joined the nanocarbon family in 2005. It was initially developed as a contrast agent for bioimaging because it can emit bright red photoluminescence from neg. charged nitrogen-vacancy centers built in the diamond matrix. A notable application of this technol. is to study the cytoplasmic dynamics of living cells by tracking single bioconjugated FNDs in intracellular medium. This article provides a crit. review on recent advances and developments of such single particle tracking (SPT) research. It summarizes SPT and related studies of FNDs in cells (such as cancer cell lines) and organisms (including zebrafish embryos, fruit fly embryos, whole nematodes, and mice) using assorted imaging techniques.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XnslWnsLc%253D&md5=049a63219eae6f145ece844bda88b540
    18. 18
      Ghanimi Fard, M.; Khabir, Z.; Reineck, P.; Cordina, N. M.; Abe, H.; Ohshima, T.; Dalal, S.; Gibson, B. C.; Packer, N. H.; Parker, L. M. Targeting cell surface glycans with lectin-coated fluorescent nanodiamonds. Nanoscale Adv. 2022, 4 (6), 1551– 1564,  DOI: 10.1039/D2NA00036A
      18
      Targeting cell surface glycans with lectin-coated fluorescent nanodiamonds
      Ghanimi Fard, Mina; Khabir, Zahra; Reineck, Philipp; Cordina, Nicole M.; Abe, Hiroshi; Ohshima, Takeshi; Dalal, Sagar; Gibson, Brant C.; Packer, Nicolle H.; Parker, Lindsay M.
      Nanoscale Advances (2022), 4 (6), 1551-1564CODEN: NAADAI; ISSN:2516-0230. (Royal Society of Chemistry)
      Glycosylation is arguably the most important functional post-translational modification in brain cells and abnormal cell surface glycan expression has been assocd. with neurol. diseases and brain cancers. In this study we developed a novel method for uptake of fluorescent nanodiamonds (FND), carbon-based nanoparticles with low toxicity and easily modifiable surfaces, into brain cell subtypes by targeting their glycan receptors with carbohydrate-binding lectins. Lectins facilitated uptake of 120 nm FND with nitrogen-vacancy centers in three types of brain cells - U87-MG astrocytes, PC12 neurons and BV-2 microglia cells. The nanodiamond/lectin complexes used in this study target glycans that have been described to be altered in brain diseases including sialic acid glycans via wheat (Triticum aestivum) germ agglutinin (WGA), high mannose glycans via tomato (Lycopersicon esculentum) lectin (TL) and core fucosylated glycans via Aleuria aurantia lectin (AAL). The lectin conjugated nanodiamonds were taken up differently by the various brain cell types with fucose binding AAL/FNDs taken up preferentially by glioblastoma phenotype astrocyte cells (U87-MG), sialic acid binding WGA/FNDs by neuronal phenotype cells (PC12) and high mannose binding TL/FNDs by microglial cells (BV-2). With increasing recognition of glycans having a role in many diseases, the lectin bioconjugated nanodiamonds developed here are well suited for further investigation into theranostic applications.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XjsVyitrg%253D&md5=1390cf7940582b1ab0f5ee79a3e0f5d5
    19. 19
      Sharmin, R.; Nusantara, A. C.; Nie, L.; Wu, K.; Elias Llumbet, A.; Woudstra, W.; Mzyk, A.; Schirhagl, R. Intracellular Quantum Sensing of Free-Radical Generation Induced by Acetaminophen (APAP) in the Cytosol, in Mitochondria and the Nucleus of Macrophages. ACS Sens. 2022, 7 (11), 3326– 3334,  DOI: 10.1021/acssensors.2c01272
      19
      Intracellular Quantum Sensing of Free-Radical Generation Induced by Acetaminophen (APAP) in the Cytosol, in Mitochondria and the Nucleus of Macrophages
      Sharmin, Rokshana; Nusantara, Anggrek C.; Nie, Linyan; Wu, Kaiqi; Elias Llumbet, Arturo; Woudstra, Willem; Mzyk, Aldona; Schirhagl, Romana
      ACS Sensors (2022), 7 (11), 3326-3334CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)
      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 resoln., these nanodiamonds were targeted to cellular locations, i.e., 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 prodn. in an organelle-specific manner, which helps predict and better understand cellular toxicity.
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    20. 20
      Terada, D.; Genjo, T.; Segawa, T. F.; Igarashi, R.; Shirakawa, M. Nanodiamonds for bioapplications–specific targeting strategies. Biochim. Biophys. Acta, Gen. Subj. 2020, 1864 (2), 129354  DOI: 10.1016/j.bbagen.2019.04.019
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      Nanodiamonds for bioapplications-specific targeting strategies
      Terada, Daiki; Genjo, Takuya; Segawa, Takuya F.; Igarashi, Ryuji; Shirakawa, Masahiro
      Biochimica et Biophysica Acta, General Subjects (2020), 1864 (2), 129354CODEN: BBGSB3; ISSN:0304-4165. (Elsevier B.V.)
      A review. Nanodiamonds (NDs) provide a unique multitasking system for drug delivery and fluorescent imaging in biol. environments. Owing to their quantum properties, NDs are expected to be employed as multifunctional probes in the future for the accurate visualization of biophys. parameters such as temp. and magnetic fields. However, the use of NDs for the selective targeting of the biomols. of interest within a complicated biol. system remains a challenge. One of the most promising solns. is the appropriate surface design of NDs based on org. chem. and biochem. The engineered NDs have high biocompatibility and dispersibility in a biol. environment and hence undergo cellular uptake through specific pathways. This review focuses on the selective targeting of NDs for biomedical and biophys. applications from the viewpoint of ND surface functionalizations and modifications. These pretreatments make possible the specific targeting of biomols. of interest on or in a cell by NDs via a designed biochem. route. The surface of NDs is covalently or noncovalently modified with silica, polymers, or biomols. to reshape them, control their size, and enhance the colloidal stability and biomol. selectivity toward the biomols. of interest. Electroporation, chem. treatment, injection, or endocytosis are the methods generally adopted to introduce NDs into living cells. The pathway, efficiency, and the cell viability depend on the selected method. In the biomedical field, the surface modification facilitates specific delivery of a drug, leading to a higher therapeutic efficacy. In biophys. applications, the surface modification paves the way for the accurate measurement of phys. parameters to gain a better understanding of various cell functions.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXht1Wqt7nI&md5=8c91af0bf9f5457c133eea889e7183f4
    21. 21
      Hemelaar, S. R.; De Boer, P.; Chipaux, M.; Zuidema, W.; Hamoh, T.; Martinez, F. P.; Nagl, A.; Hoogenboom, J. P.; Giepmans, B. N. G.; Schirhagl, R. Nanodiamonds as multi-purpose labels for microscopy. Sci. Rep. 2017, 7 (1), 720  DOI: 10.1038/s41598-017-00797-2
      21
      Nanodiamonds as multi-purpose labels for microscopy
      Hemelaar S R; Chipaux M; Hamoh T; Martinez F Perona; Nagl A; Schirhagl R; de Boer P; Giepmans B N G; Zuidema W; Hoogenboom J P
      Scientific reports (2017), 7 (1), 720 ISSN:.
      Nanodiamonds containing fluorescent nitrogen-vacancy centers are increasingly attracting interest for use as a probe in biological microscopy. This interest stems from (i) strong resistance to photobleaching allowing prolonged fluorescence observation times; (ii) the possibility to excite fluorescence using a focused electron beam (cathodoluminescence; CL) for high-resolution localization; and (iii) the potential use for nanoscale sensing. For all these schemes, the development of versatile molecular labeling using relatively small diamonds is essential. Here, we show the direct targeting of a biological molecule with nanodiamonds as small as 70 nm using a streptavidin conjugation and standard antibody labelling approach. We also show internalization of 40 nm sized nanodiamonds. The fluorescence from the nanodiamonds survives osmium-fixation and plastic embedding making them suited for correlative light and electron microscopy. We show that CL can be observed from epon-embedded nanodiamonds, while surface-exposed nanoparticles also stand out in secondary electron (SE) signal due to the exceptionally high diamond SE yield. Finally, we demonstrate the magnetic read-out using fluorescence from diamonds prior to embedding. Thus, our results firmly establish nanodiamonds containing nitrogen-vacancy centers as unique, versatile probes for combining and correlating different types of microscopy, from fluorescence imaging and magnetometry to ultrastructural investigation using electron microscopy.
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    22. 22
      McGuinness, L. P.; Yan, Y.; Stacey, A.; Simpson, D. A.; Hall, L. T.; Maclaurin, D.; Prawer, S.; Mulvaney, P.; Wrachtrup, J.; Caruso, F.; Scholten, R. E.; Hollenberg, L. C. L. Quantum measurement and orientation tracking of fluorescent nanodiamonds inside living cells. Nat. Nanotechnol. 2011, 6 (6), 358– 363,  DOI: 10.1038/nnano.2011.64
      22
      Quantum measurement and orientation tracking of fluorescent nanodiamonds inside living cells
      McGuinness, L. P.; Yan, Y.; Stacey, A.; Simpson, D. A.; Hall, L. T.; MacLaurin, D.; Prawer, S.; Mulvaney, P.; Wrachtrup, J.; Caruso, F.; Scholten, R. E.; Hollenberg, L. C. L.
      Nature Nanotechnology (2011), 6 (6), 358-363CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
      Fluorescent particles are routinely used to probe biol. processes. The quantum properties of single spins within fluorescent particles have been explored in the field of nanoscale magnetometry, but not yet in biol. environments. Here, the authors demonstrate optically detected magnetic resonance of individual fluorescent nanodiamond nitrogen-vacancy centers inside living human HeLa cells, and measure their location, orientation, spin levels and spin coherence times with nanoscale precision. Quantum coherence was measured through Rabi and spin-echo sequences over long (>10 h) periods, and orientation was tracked with effective 1° angular precision over acquisition times of 89 ms. The quantum spin levels served as fingerprints, allowing individual centers with identical fluorescence to be identified and tracked simultaneously. Furthermore, monitoring decoherence rates in response to changes in the local environment may provide new information about intracellular processes. The expts. reported here demonstrate the viability of controlled single spin probes for nanomagnetometry in biol. systems, opening up a host of new possibilities for quantum-based imaging in the life sciences.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXntVynsLY%253D&md5=38b18ea65726303e8f01039f13bb6514
    23. 23
      Neumann, P.; Jakobi, I.; Dolde, F.; Burk, C.; Reuter, R.; Waldherr, G.; Honert, J.; Wolf, T.; Brunner, A.; Shim, J. H.; Suter, D. High-precision nanoscale temperature sensing using single defects in diamond. Nano Lett. 2013, 13 (6), 2738– 2742,  DOI: 10.1021/nl401216y
      23
      High-Precision Nanoscale Temperature Sensing Using Single Defects in Diamond
      Neumann, P.; Jakobi, I.; Dolde, F.; Burk, C.; Reuter, R.; Waldherr, G.; Honert, J.; Wolf, T.; Brunner, A.; Shim, J. H.; Suter, D.; Sumiya, H.; Isoya, J.; Wrachtrup, J.
      Nano Letters (2013), 13 (6), 2738-2742CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
      Measuring local temp. with a spatial resoln. on the order of a few nanometers has a wide range of applications in the semiconductor industry and in material and life sciences. For example, probing temp. on the nanoscale with high precision can potentially be used to detect small, local temp. changes like those caused by chem. reactions or biochem. processes. However, precise nanoscale temp. measurements have not been realized so far owing to the lack of adequate probes. The authors exptl. demonstrate a novel nanoscale temp. sensing technique based on optically detected ESR in single at. defects in diamonds. These diamond sensor sizes range from a micrometer down to a few tens of nanometers. The authors achieve a temp. noise floor of 5 mK/Hz1/2 for single defects in bulk sensors. Using doped nano-diamonds as sensors, the temp. noise floor is 130 mK/Hz1/2 and accuracies down to 1 mK for nanocrystal sizes and therefore length scales of a few tens of nanometers. This combination of precision and position resoln., combined with the outstanding sensor photostability, should allow the measurement of the heat produced by chem. interactions involving a few or single mols. even in heterogeneous environments like cells.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXosVOhsLg%253D&md5=0670fe270d927f519d6fd05b64a9e80b
    24. 24
      Kucsko, G.; Maurer, P. C.; Yao, N. Y.; Kubo, M.; Noh, H. J.; Lo, P. K.; Park, H.; Lukin, M. D. Nanometre-scale thermometry in a living cell. Nature 2013, 500 (7460), 54– 58,  DOI: 10.1038/nature12373
      24
      Nanometre-scale thermometry in a living cell
      Kucsko, G.; Maurer, P. C.; Yao, N. Y.; Kubo, M.; Noh, H. J.; Lo, P. K.; Park, H.; Lukin, M. D.
      Nature (London, United Kingdom) (2013), 500 (7460), 54-58CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Sensitive probing of temp. variations on nanometer scales is an outstanding challenge in many areas of modern science and technol. In particular, a thermometer capable of subdegree temp. resoln. over a large range of temps. as well as integration within a living system could provide a powerful new tool in many areas of biol., phys. and chem. research. Possibilities range from the temp.-induced control of gene expression and tumor metab. to the cell-selective treatment of disease and the study of heat dissipation in integrated circuits. By combining local light-induced heat sources with sensitive nanoscale thermometry, it may also be possible to engineer biol. processes at the subcellular level. Here the authors demonstrate a new approach to nanoscale thermometry that uses coherent manipulation of the electronic spin assocd. with nitrogen-vacancy color centers in diamond. The authors' technique makes it possible to detect temp. variations as small as 1.8 mK (a sensitivity of 9 mK Hz-1/2) in an ultrapure bulk diamond sample. Using nitrogen-vacancy centers in diamond nanocrystals (nanodiamonds), the authors directly measure the local thermal environment on length scales as short as 200 nm. Finally, by introducing both nanodiamonds and gold nanoparticles into a single human embryonic fibroblast, the authors demonstrate temp.-gradient control and mapping at the subcellular level, enabling unique potential applications in life sciences.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXht1SjtbfJ&md5=bb54bbec1542e1ee2ac19fa63a8c5845
    25. 25
      Tian, Y.; Nusantara, A. C.; Hamoh, T.; Mzyk, A.; Tian, X.; Perona Martinez, F.; Li, R.; Permentier, H. P.; Schirhagl, R. Functionalized fluorescent nanodiamonds for simultaneous drug delivery and quantum sensing in HeLa cells. ACS Appl. Mater. Interfaces 2022, 14 (34), 39265– 39273,  DOI: 10.1021/acsami.2c11688
      25
      Functionalized Fluorescent Nanodiamonds for Simultaneous Drug Delivery and Quantum Sensing in HeLa Cells
      Tian, Yuchen; Nusantara, Anggrek C.; Hamoh, Thamir; Mzyk, Aldona; Tian, Xiaobo; Perona Martinez, Felipe; Li, Runrun; Permentier, Hjalmar P.; Schirhagl, Romana
      ACS Applied Materials & Interfaces (2022), 14 (34), 39265-39273CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)
      Here, we present multifunctional fluorescent nanodiamonds (FNDs) for simultaneous drug delivery and free radical detection. For this purpose, we modified FNDs contg. nitrogen vacancy (NV) centers with a diazoxide deriv. We found that our particles enter cells more easily and are able to deliver this cancer drug into HeLa cells. The particles were characterized by IR spectroscopy, dynamic light scattering, and secondary electron microscopy. Compared to the free drug, we observe a sustained release over 72 h rather than 12 h for the free drug. Apart from releasing the drug, with these particles, we can measure the drug's effect on free radical generation directly. This has the advantage that the response is measured locally, where the drug is released. These FNDs change their optical properties based on their magnetic surrounding. More specifically, we make use of a technique called relaxometry to detect spin noise from the free radical at the nanoscale with subcellular resoln. We further compared the results from our new technique with a conventional fluorescence assay for the detection of reactive oxygen species. This provides a new method to investigate the relationship between drug release and the response by the cell via radical formation or inhibition.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XitFGhtbjF&md5=d496a2bb9836b6dc464080555ad16805
    26. 26
      Norouzi, N.; Nusantara, A. C.; Ong, Y.; Hamoh, T.; Nie, L.; Morita, A.; Zhang, Y.; Mzyk, A.; Schirhagl, R. Relaxometry for detecting free radical generation during Bacteria’s response to antibiotics. Carbon 2022, 199, 444– 452,  DOI: 10.1016/j.carbon.2022.08.025
      26
      Relaxometry for detecting free radical generation during Bacteria's response to antibiotics
      Norouzi, Neda; Nusantara, Anggrek Citra; Ong, Yori; Hamoh, Thamir; Nie, Linyan; Morita, Aryan; Zhang, Yue; Mzyk, Aldona; Schirhagl, Romana
      Carbon (2022), 199 (), 444-452CODEN: CRBNAH; ISSN:0008-6223. (Elsevier Ltd.)
      Free radical generation plays a key role in killing bacteria by antibiotics. However, radicals are short-lived and reactive, and thus difficult to detect for the state of the art. Here we use a technique which allows optical nanoscale magnetic resonance imaging (MRI) to detect radical generation on the scale of single bacteria. We demonstrate that the radical generation in Staphylococcus aureus increases in the presence of UV irradn. as well as vancomycin and is dependent on the antibiotics dose. With a method based on ensembles of nitrogen vacancy (NV) centers in diamond, we were able to follow the radical formation near individual bacteria over the whole duration of the expt. to reveal the dynamics of radical generation. Using this new approach, we obsd. free radical concns. within nanoscale voxels around the diamond particles and detd. its exact timing depending on the antibiotic dose. Since changes in the response to antibiotics emerge in only a few bacteria of the entire population, such a single-cell approach can prove highly valuable for research into drug resistance.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XitF2ju7fM&md5=66e476e7cef542049adc40bccb487cad
    27. 27
      Wu, K.; Nie, L.; Nusantara, A. C.; Woudstra, W.; Vedelaar, T.; Sigaeva, A.; Schirhagl, R. Diamond Relaxometry as a Tool to Investigate the Free Radical Dialogue between Macrophages and Bacteria. ACS Nano 2023, 17 (2), 1100– 1111,  DOI: 10.1021/acsnano.2c08190
      27
      Diamond Relaxometry as a Tool to Investigate the Free Radical Dialogue between Macrophages and Bacteria
      Wu, Kaiqi; Nie, Linyan; Nusantara, Anggrek C.; Woudstra, Willem; Vedelaar, Thea; Sigaeva, Alina; Schirhagl, Romana
      ACS Nano (2023), 17 (2), 1100-1111CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
      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 obsd. 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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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXnsVaitg%253D%253D&md5=f73deecbc2a649f7c3bc48dfc61f836c
    28. 28
      Reyes-San-Martin, C.; Hamoh, T.; Zhang, Y.; Berendse, L.; Klijn, C.; Li, R.; Llumbet, A. E.; Sigaeva, A.; Kawałko, J.; Mzyk, A.; Schirhagl, R. Nanoscale mri for selective labeling and localized free radical measurements in the acrosomes of single sperm cells. ACS Nano 2022, 16 (7), 10701– 10710,  DOI: 10.1021/acsnano.2c02511
      28
      Nanoscale MRI for Selective Labeling and Localized Free Radical Measurements in the Acrosomes of Single Sperm Cells
      Reyes-San-Martin, Claudia; Hamoh, Thamir; Zhang, Yue; Berendse, Lotte; Klijn, Carline; Li, Runrun; Llumbet, Arturo E.; Sigaeva, Alina; Kawalko, Jakub; Mzyk, Aldona; Schirhagl, Romana
      ACS Nano (2022), 16 (7), 10701-10710CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
      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.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xhs1elu77M&md5=287c8abe16a0fa344d4e0f7b181f3030
    29. 29
      Shenderova, O. A.; Shames, A. I.; Nunn, N. A.; Torelli, M. D.; Vlasov, I.; Zaitsev, A. Synthesis, properties, and applications of fluorescent diamond particles. J. Vac. Sci. Technol., B: Nanotechnol. Microelectron.: Mater., Process., Meas., Phenom. 2019, 37 (3), 030802  DOI: 10.1116/1.5089898
      29
      Review Article: Synthesis, properties, and applications of fluorescent diamond particles
      Shenderova, Olga A.; Shames, Alexander I.; Nunn, Nicholas A.; Torelli, Marco D.; Vlasov, Igor; Zaitsev, Alexander
      Journal of Vacuum Science & Technology, B: Nanotechnology & Microelectronics: Materials, Processing, Measurement, & Phenomena (2019), 37 (3), 030802/1-030802/27CODEN: JVSTCN; ISSN:2166-2746. (American Institute of Physics)
      A review. Diamond particles contg. color centers-fluorescent crystallog. defects embedded within the diamond lattice-outperform other classes of fluorophores by providing a combination of unmatched photostability, intriguing coupled magneto-optical properties, intrinsic biocompatibility, and outstanding mech. and chem. robustness. This exceptional combination of properties positions fluorescent diamond particles as unique fluorophores with emerging applications in a variety of fields, including bioimaging, ultrasensitive metrol. at the nanoscale, fluorescent tags in industrial applications, and even potentially as magnetic resonance imaging contrast agents. However, prodn. of fluorescent nanodiamond (FND) is nontrivial, since it requires irradn. with high-energy particles to displace carbon atoms and create vacancies-a primary constituent in the majority color centers. In this review, centrally focused on material developments, major steps of FND prodn. are discussed with emphasis on current challenges in the field and possible solns. The authors demonstrate how the combination of fluorescent spectroscopy and ESR provides valuable insight into the types of radiation-induced defects formed and their evolution upon thermal annealing, thereby guiding FND performance optimization. A recent breakthrough process allowing for prodn. of fluorescent diamond particles with vibrant blue, green, and red fluorescence is also discussed. Finally, the authors conclude with demonstrations of a few FND applications in the life science arena and in industry. (c) 2019 American Institute of Physics.
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    30. 30
      Ong, S. Y.; Van Harmelen, R. J. J.; Norouzi, N.; Offens, F.; Venema, I. M.; Najafi, M. H.; Schirhagl, R. Interaction of nanodiamonds with bacteria. Nanoscale 2018, 10 (36), 17117– 17124,  DOI: 10.1039/C8NR05183F
      30
      Interaction of nanodiamonds with bacteria
      Ong, S. Y.; van Harmelen, R. J. J.; Norouzi, N.; Offens, F.; Venema, I. M.; Habibi Najafi, M. B.; Schirhagl, R.
      Nanoscale (2018), 10 (36), 17117-17124CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)
      Nanocarbons come in many forms and among their applications is the engineering of biocompatible and antibacterial materials. Studies have shown that diamond nanoparticles might have the interesting combination of both properties: they are highly biocompatible, while surprisingly reducing bacterial viability or growth at the same time. In this article, we consider for the first time the interaction of milled HPHT nanodiamonds with bacteria. These nanoparticles are capable of hosting nitrogen-vacancy (NV) centers, which provide stable fluorescence with potential use in sensing applications. An initial study was performed to assess the interaction of partially oxidized monocryst. nanodiamonds with Gram pos. S. aureus ATCC 12600 and Gram neg. E. coli ATCC 8739. It was shown that for S. aureus ATCC 12600, the presence of these nanodiamonds leads to a sharp redn. of colony forming ability under optimal conditions. A different effect was obsd. on Gram neg. E. coli ATCC 8739, where no significant adverse effects of ND presence was obsd. The mode of interaction was further studied by electron microscopy and confocal microscopy. The effects of NDs on S. aureus viability were found to depend on many factors, including the concn. and size of nanoparticles, the suspension medium and incubation time.
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    31. 31
      Jaiswal, J.; Gupta, S. K.; Kreuter, J. Preparation of biodegradable cyclosporine nanoparticles by high-pressure emulsification-solvent evaporation process. J. Controlled Release 2004, 96 (1), 169– 178,  DOI: 10.1016/j.jconrel.2004.01.017
      31
      Preparation of biodegradable cyclosporine nanoparticles by high-pressure emulsification-solvent evaporation process
      Jaiswal, Jagdish; Gupta, Suresh Kumar; Kreuter, Jorg
      Journal of Controlled Release (2004), 96 (1), 169-178CODEN: JCREEC; ISSN:0168-3659. (Elsevier)
      The cyclic endecapeptide cyclosporine (CsA), a potent immunosuppressive drug, was incorporated into biodegradable poly (DL-lactide-co-glycolide) (dl-PLG) 50/50, 65/35 and PEG 5000-70/30 dL-PLG to improve the oral bioavailability and pharmacokinetics. Nanoparticles were prepd. by a high-pressure emulsification-solvent evapn. (HPESE) process. The CsA-loaded nanoparticles were evaluated for particle size, zeta potential, surface morphol. by SEM, thermal characterizations by differential scanning calorimetry (DSC), encapsulation efficiency (E.E.%) and in vitro release. The amt. of CsA loaded into the nanoparticles was detd. using high-performance liq. chromatog. (HPLC) at a detection wavelength of 210 nm. The mobile phase was acetonitrile-water (70:30% vol./vol.) and flow rate was set at 1.5 mL min-1. The photon correlation spectroscopy showed that the particles size were <250 nm and polydispersity index (PI) <0.14. The zeta potential was pos. for 200 mg and neg. for 400 mg of polymer compn., resp. The SEM micrographs revealed that the nanoparticles were spherical and smooth. The drug loading was between 82% and 92%. Differential scanning calorimetry (DSC) studies did not show the melting endotherm for CsA in the drug-loaded nanoparticles. In-vitro release in intestinal fluid pH 6.8 (USP XXIV) showed a cumulative percent release of 30-45% CsA in 8 h. The physicochem. properties showed that the dl-PLG and PEG-DLPLG nanoparticles could be an effective carrier for oral CsA delivery. The reported method is easy, reproducible and can be automated for batch scale prodn.
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    32. 32
      de Graaf, I. A. M.; Olinga, P.; De Jager, M. H.; Merema, M. T.; De Kanter, R.; Van De Kerkhof, E. G.; Groothuis, G. M. Preparation and incubation of precision-cut liver and intestinal slices for application in drug metabolism and toxicity studies. Nat. Protoc. 2010, 5 (9), 1540– 1551,  DOI: 10.1038/nprot.2010.111
      32
      Preparation and incubation of precision-cut liver and intestinal slices for application in drug metabolism and toxicity studies
      de Graaf, Inge A. M.; Olinga, Peter; de Jager, Marina H.; Merema, Marjolijn T.; de Kanter, Ruben; van de Kerkhof, Esther G.; Groothuis, Geny M. M.
      Nature Protocols (2010), 5 (9), 1540-1551CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)
      Precision-cut tissue slices (PCTS) are viable ex vivo explants of tissue with a reproducible, well defined thickness. They represent a mini-model of the organ under study and contain all cells of the tissue in their natural environment, leaving intercellular and cell-matrix interactions intact, and are therefore highly appropriate for studying multicellular processes. PCTS are mainly used to study the metab. and toxicity of xenobiotics, but they are suitable for many other purposes. Here we describe the protocols to prep. and incubate rat and human liver and intestinal slices. Slices are prepd. from fresh liver by making a cylindrical core using a drill with a hollow bit, from which slices are cut with a specially designed tissue slicer. Intestinal tissue is embedded in cylinders of agarose before slicing. Slices remain viable for 24 h (intestine) and up to 96 h (liver) when incubated in 6- or 12-well plates under 95% O2/5% CO2 atm.
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    33. 33
      Foroozandeh, P.; Aziz, A. A. Insight into cellular uptake and intracellular trafficking of nanoparticles. Nanoscale Res. Lett. 2018, 13, 339,  DOI: 10.1186/s11671-018-2728-6
      33
      Insight into Cellular Uptake and Intracellular Trafficking of Nanoparticles
      Foroozandeh Parisa; Aziz Azlan Abdul; Aziz Azlan Abdul
      Nanoscale research letters (2018), 13 (1), 339 ISSN:1931-7573.
      Nanoparticle science is rapidly changing the landscape of various scientific fields and defining new technological platforms. This is perhaps even more evident in the field of nanomedicine whereby nanoparticles have been used as a tool for the treatment and diagnosis of many diseases. However, despite the tremendous benefit conferred, common pitfalls of this technology is its potential short and long-term effects on the human body. To understand these issues, many scientific studies have been carried out. This review attempts to shed light on some of these studies and its outcomes. The topics that were examined in this review include the different possible uptake pathways of nanoparticles and intracellular trafficking routes. Additionally, the effect of physicochemical properties of nanoparticle such as size, shape, charge and surface chemistry in determining the mechanism of uptake and biological function of nanoparticles are also addressed.
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    34. 34
      van der Laan, K. J.; Naulleau, J.; Damle, V. G.; Sigaeva, A.; Jamot, N.; Perona-Martinez, F. P.; Chipaux, M.; Schirhagl, R. Toward using fluorescent nanodiamonds to study chronological aging in Saccharomyces cerevisiae. Anal. Chem. 2018, 90 (22), 13506– 13513,  DOI: 10.1021/acs.analchem.8b03431
      34
      Towards Using Fluorescent Nanodiamonds To Study Chronological Aging in Saccharomyces cerevisiae
      van der Laan, Kiran J.; Naulleau, Julie; Damle, Viraj G.; Sigaeva, Alina; Jamot, Nicolas; Perona-Martinez, Felipe P.; Chipaux, Mayeul; Schirhagl, Romana
      Analytical Chemistry (Washington, DC, United States) (2018), 90 (22), 13506-13513CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      One of the theories aiming to explain cellular aging is the free radical theory of aging, which describes the possible role of increased prodn. 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 mol. 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 chronol. 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.
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    35. 35
      Vaijayanthimala, V.; Tzeng, Y. K.; Chang, H. C.; Li, C. L. The biocompatibility of fluorescent nanodiamonds and their mechanism of cellular uptake. Nanotechnology 2009, 20 (42), 425103  DOI: 10.1088/0957-4484/20/42/425103
      35
      The biocompatibility of fluorescent nanodiamonds and their mechanism of cellular uptake
      Vaijayanthimala, Vairakkannu; Tzeng, Yan-Kai; Chang, Huan-Cheng; Li, Chung-Leung
      Nanotechnology (2009), 20 (42), 425103/1-425103/9CODEN: NNOTER; ISSN:1361-6528. (Institute of Physics Publishing)
      The labeling of cells with fluorescent nanoparticles is promising for various biomedical applications. The objective of this study is to evaluate the biocompatibility and the mechanism of the cellular uptake of fluorescent nanodiamonds (FNDs) in cancer cells (HeLa) and pre-adipocytes (3T3-L1). With flow cytometry and the use of a battery of metabolic and cytoskeletal inhibitors, we found that the mechanism of the FND uptake in both cells is by energy-dependent clathrin-mediated endocytosis. In addn., the surface charge of FND influences its cellular uptake, as the uptake of poly-L-lysine-coated FNDs is better than that of oxidative-acid-purified FNDs at the same concn. in regular medium with or without serum. We also confirm that the proliferative potential of FND-treated and untreated cells does not exhibit any significant differences when measured at bulk cultures, and more stringently at clonal cell d. Further biocompatibility studies indicate that the in vitro differentiation of 3T3-L1 pre-adipocytes and 489-2 osteoprogenitors is not affected by the FND treatment. Our results show that FNDs are biocompatible and ideal candidates for potential applications in human stem cell research.
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    36. 36
      Hemelaar, S. R.; Saspaanithy, B.; L’hommelet, S. R.; Perona Martinez, F. P.; Van der Laan, K. J.; Schirhagl, R. The response of HeLa cells to fluorescent nanodiamond uptake. Sensors 2018, 18 (2), 355,  DOI: 10.3390/s18020355
      36
      The response of hela cells to fluorescent nanodiamond uptake
      Hemelaar, Simon R.; Saspaanithy, Babujhi; L'Hommelet, Severin R. M.; Martinez, Felipe P. Perona; van der Laan, Kiran J.; Schirhagl, Romana
      Sensors (2018), 18 (2), 355/1-355/15CODEN: SENSC9; ISSN:1424-8220. (MDPI AG)
      Fluorescent nanodiamonds are promising probes for nanoscale magnetic resonance measurements. Their phys. properties predict them to have particularly useful applications in intracellular anal. Before using them in intracellular expts. however, it should be clear whether diamond particles influence cell biol. While cytotoxicity has already been ruled out in previous studies, we consider the non-fatal influence of fluorescent nanodiamonds on the formation of reactive oxygen species (an important stress indicator and potential target for intracellular sensing) for the first time. We investigated the influence of different sizes, shapes and concns. of nanodiamonds on the genetic and protein level involved in oxidative stress-related pathways of the HeLa cell, an important model cell line in research. The changes in viability of the cells and the difference in intracellular levels of free radicals, after diamond uptake, are surprisingly small. At lower diamond concns., the cellular metab. cannot be distinguished from that of untreated cells. This research supports the claims of non-toxicity and includes less obvious non-fatal responses. Finally, we give a handhold concerning the diamond concn. and size to use for non-toxic, intracellular measurements in favor of (cancer) research in HeLa cells.
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    37. 37
      Zipfel, W. R.; Williams, R. M.; Christie, R.; Nikitin, A. Y.; Hyman, B. T.; Webb, W. W. Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation. Proc. Natl. Acad. Sci. U.S.A. 2003, 100 (12), 7075– 7080,  DOI: 10.1073/pnas.0832308100
      37
      Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation
      Zipfel, Warren R.; Williams, Rebecca M.; Christie, Richard; Nikitin, Alexander Yu; Hyman, Bradley T.; Webb, Watt W.
      Proceedings of the National Academy of Sciences of the United States of America (2003), 100 (12), 7075-7080CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Multicolor nonlinear microscopy of living tissue using two- and three-photon-excited intrinsic fluorescence combined with second harmonic generation by supermol. structures produces images with the resoln. and detail of std. histol. without the use of exogenous stains. Imaging of intrinsic indicators within tissue, such as NAD, retinol, indoleamines, and collagen provides crucial information for physiol. and pathol. The efficient application of multiphoton microscopy to intrinsic imaging requires knowledge of the nonlinear optical properties of specific cell and tissue components. Here we compile and demonstrate applications involving a range of intrinsic mols. and mol. assemblies that enable direct visualization of tissue morphol., cell metab., and disease states such as Alzheimer's disease and cancer.
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    38. 38
      Jun, Y. W.; Kim, H. R.; Reo, Y. J.; Dai, M.; Ahn, K. H. Addressing the autofluorescence issue in deep tissue imaging by two-photon microscopy: the significance of far-red emitting dyes. Chem. Sci. 2017, 8 (11), 7696– 7704,  DOI: 10.1039/C7SC03362A
      38
      Addressing the autofluorescence issue in deep tissue imaging by two-photon microscopy: the significance of far-red emitting dyes
      Jun, Yong Woong; Kim, Hye Rim; Reo, Ye Jin; Dai, Mingchong; Ahn, Kyo Han
      Chemical Science (2017), 8 (11), 7696-7704CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      The fluorescence imaging of tissue is essential for studying biol. events beyond the cellular level. Two-photon microscopy based on the nonlinear light absorption of fluorescent dyes is a viable tool for the high resoln. imaging of tissue. A key limitation for deep tissue imaging is the autofluorescence from intrinsic biomols. Here, we report a systematic study that discloses relative autofluorescence interference, which is dependent on the type of tissue and the excitation and emission wavelengths in two-photon imaging. Among the brain, kidney, liver, lung, and spleen mouse tissues examd., the kidney tissue exhibited prominent autofluorescence followed by the liver and others. Notably, regardless of the tissue type, prominent autofluorescence is obsd. not only from the green emission channel but also from the yellow emission channel where common two-photon absorbing dyes also emit, whereas there is minimal autofluorescence from the red channel. The autofluorescence is slightly influenced by the excitation wavelength. Toward minimal autofluorescence, we developed a new class of two-photon absorbing dyes that are far-red emitting, water-sol., and very bright inside cells as well as in tissue. A comparative assessment of the imaging depth, which is dependent on the three selected dyes that emit in the blue-green, yellow, and far-red regions, shows the importance of far-red emitting dyes for deep tissue imaging.
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    39. 39
      Li, R.; Vedelaar, T.; Mzyk, A.; Morita, A.; Padamati, S. K.; Schirhagl, R. Following Polymer Degradation with Nanodiamond Magnetometry. ACS Sens. 2022, 7 (1), 123– 130,  DOI: 10.1021/acssensors.1c01782
      39
      Following Polymer Degradation with Nanodiamond Magnetometry
      Li, Runrun; Vedelaar, Thea; Mzyk, Aldona; Morita, Aryan; Padamati, Sandeep Kumar; Schirhagl, Romana
      ACS Sensors (2022), 7 (1), 123-130CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)
      Degradable polymers are widely used in the biomedical fields due to non-toxicity and great biocompatibility and biodegradability, and it is crucial to understand how they degrade. These polymers are exposed to various biochem. media in medical practice. Hence, it is important to precisely follow the degrdn. of the polymer in real time. In this study, we made use of diamond magnetometry for the first time to track polymer degrdn. with nanoscale precision. The method is based on a fluorescent defect in nanodiamonds, which changes its optical properties based on its magnetic surrounding. Since optical signals can be read out more sensitively than magnetic signals, this method allows unprecedented sensitivity. We used a specific mode of diamond magnetometry called relaxometry or T1 measurements. These are sensitive to magnetic noise and thus can detect paramagnetic species (gadolinium in this case). Nanodiamonds were incorporated into polylactic acid (PLA) films and PLA nanoparticles in order to follow polymer degrdn. However, in principle, they can be incorporated into other polymers too. We found that T1 consts. decreased gradually with the erosion of the film exposed to an alk. condition. In addn., the mobility of nanodiamonds increased, which allows us to est. polymer viscosity. The degrdn. rates obtained using this approach were in good agreement with data obtained by quartz crystal microbalance, Fourier-transform IR spectroscopy, and at. force microscopy.
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    40. 40
      Prabhakar, N.; Khan, M. H.; Peurla, M.; Chang, H. C.; Hänninen, P. E.; Rosenholm, J. M. Intracellular trafficking of fluorescent nanodiamonds and regulation of their cellular toxicity. ACS Omega 2017, 2 (6), 2689– 2693,  DOI: 10.1021/acsomega.7b00339
      40
      Intracellular Trafficking of Fluorescent Nanodiamonds and Regulation of Their Cellular Toxicity
      Prabhakar, Neeraj; Khan, Meraj H.; Peurla, Markus; Chang, Huan-Cheng; Hanninen, Pekka E.; Rosenholm, Jessica M.
      ACS Omega (2017), 2 (6), 2689-2693CODEN: ACSODF; ISSN:2470-1343. (American Chemical Society)
      Cellular management of fluorescent nanodiamonds (FNDs) has been studied for better understanding in designing potential applications of FNDs in biomedicine. The FNDs have been shown to be photostable probes for bioimaging and thus are well suited for e.g. long term tracking purposes. FNDs also exhibit good biocompatibility and in general, low toxicity for cell labeling. To demonstrate the underlying mechanism of cells coping the low but potentially toxic effects by non-degradable FNDs, the authors have studied their temporal intracellular trafficking. FNDs are localized as distinct populations inside cells in early endosomes, lysosomes, and in proximity to the plasma membrane. The localization of FNDs in early endosomes suggests the internalization of FNDs, and lysosomal localization in turn can be interpreted as a pre-state for exocytosis via lysosomal degrdn. pathway. The endocytosis and exocytosis appears to be occurring simultaneously in the authors' observations. The mechanism of continuous endocytosis and exocytosis of FNDs could be necessary for cells to maintain normal proliferation. Further, a 120 h cell growth assay was performed to verify the long-term biocompatibility of FNDs for cellular studies.
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    41. 41
      Nie, L.; Zhang, Y.; Li, L.; van Rijn, P.; Schirhagl, R. pH sensitive dextran coated fluorescent nanodiamonds as a biomarker for hela cells endocytic pathway and increased cellular uptake. Nanomaterials 2021, 11 (7), 1837,  DOI: 10.3390/nano11071837
      41
      The pH Sensitive Dextran Coated Fluorescent Nanodiamonds as a Biomarker for HeLa Cells Endocytic Pathway and Increased Cellular Uptake
      Nie, Linyan; Zhang, Yue; Li, Lei; van Rijn, Patrick; Schirhagl, Romana
      Nanomaterials (2021), 11 (7), 1837CODEN: NANOKO; ISSN:2079-4991. (MDPI AG)
      Fluorescent nanodiamonds are a useful for biosensing of intracellular signaling networks or environmental changes (such as temp., pH or free radical generation). HeLa cells are interesting to study with these nanodiamonds since they are a model cell system that is widely used to study cancer-related diseases. However, they only internalize low nos. of nanodiamond particles very slowly via the endocytosis pathway. In this work, we show that pH-sensitive, dextran-coated fluorescent nanodiamonds can be used to visualize this pathway. Addnl., this coating improved diamond uptake in HeLa cells by 5.3 times (*** p < 0.0001) and decreased the required time for uptake to only 30 min. We demonstrated further that nanodiamonds enter HeLa cells via endolysosomes and are eventually expelled by cells.
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    42. 42
      Selby, L. I.; Cortez-Jugo, C. M.; Such, G. K.; Johnston, A. P. Nanoescapology: progress toward understanding the endosomal escape of polymeric nanoparticles. Wiley Interdiscip. Rev.: Nanomed. Nanobiotechnol. 2017, 9 (5), e1452  DOI: 10.1002/wnan.1452
      There is no corresponding record for this reference.
    43. 43
      Sigaeva, A.; Hochstetter, A.; Bouyim, S.; Chipaux, M.; Stejfova, M.; Cigler, P.; Schirhagl, R. Single-Particle Tracking and Trajectory Analysis of Fluorescent Nanodiamonds in Cell-Free Environment and Live Cells. Small 2022, 18 (39), 2201395  DOI: 10.1002/smll.202201395
      43
      Single-Particle Tracking and Trajectory Analysis of Fluorescent Nanodiamonds in Cell-Free Environment and Live Cells
      Sigaeva, Alina; Hochstetter, Axel; Bouyim, Sighom; Chipaux, Mayeul; Stejfova, Miroslava; Cigler, Petr; Schirhagl, Romana
      Small (2022), 18 (39), 2201395CODEN: SMALBC; ISSN:1613-6810. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Diamond magnetometry can provide new insights on the prodn. of free radicals inside live cells due to its high sensitivity and spatial resoln. However, the measurements often lack intracellular context for the recorded signal. In this paper, the possible use of single-particle tracking and trajectory anal. of fluorescent nanodiamonds (FNDs) to bridge that gap is explored. It starts with simulating a set of different possible scenarios of a particles movement, reflecting different modes of motion, degrees of confinement, as well as shapes and sizes of that confinement. Then, the insights from the anal. of the simulated trajectories are applied to describe the movement of FNDs in glycerol solns. It is shown that the measurements are in good agreement with the previously reported findings and that trajectory anal. 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.
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  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.3c01452.

    • Protocols for nanodiamond tracking; characterization of PLA-FNDs, including SEM, DLS, confocal images; tracking results; XTT assay results to confirm the biocompatibility of particles; and details on quantitative image analysis (PDF)


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