Nanodiamond uptake in colon cancer cells: the influence of direction and trypsin-EDTA treatment.

Nanoparticles are routinely used in cell biology. They deliver drugs or function as labels or sensors. For many of these applications it is essential that the nanoparticles enter the cells. While some cell types readily ingest all kinds of particles, others just don't. We report that uptake can be enhanced for some cells if the particles are administered from the basolateral side of the cells (in this case from below). Compared to apical uptake (from above), we report an 8-fold increase in the number of fluorescent nanodiamonds internalized by the colon cancer cell line HT29. Up to 96% of the cells treated by a modified protocol contain at least one nanodiamond, whereas in the control group we could observe nanodiamonds in less than half of the cells. We were also able to show that simple treatment of cell clusters with trypsin-EDTA leads to the same enhancement of the nanodiamond uptake as seeding the cells on top of the nanoparticles. Although our study is focused on nanodiamonds in HT29 cells, we believe that this method could also be applicable for other nanoparticles and cells with a specific directionality.

Nanodiamonds and Their Applications in Cells.

Diamonds owe their fame to a unique set of outstanding properties. They combine a high refractive index, hardness, great stability and inertness, and low electrical but high thermal conductivity. Diamond defects have recently attracted a lot of attention. Given this unique list of properties, it is not surprising that diamond nanoparticles are utilized for numerous applications. Due to their hardness, they are routinely used as abrasives. Their small and uniform size qualifies them as attractive carriers for drug delivery. The stable fluorescence of diamond defects allows their use as stable single photon sources or biolabels. The magnetic properties of the defects make them stable spin qubits in quantum information. This property also allows their use as a sensor for temperature, magnetic fields, electric fields, or strain. This Review focuses on applications in cells. Different diamond materials and the special requirements for the respective applications are discussed. Methods to chemically modify the surface of diamonds and the different hurdles one has to overcome when working with cells, such as entering the cells and biocompatibility, are described. Finally, the recent developments and applications in labeling, sensing, drug delivery, theranostics, antibiotics, and tissue engineering are critically discussed.

The Response of HeLa Cells to Fluorescent NanoDiamond Uptake.

Fluorescent nanodiamonds are promising probes for nanoscale magnetic resonance measurements. Their physical properties predict them to have particularly useful applications in intracellular analysis. Before using them in intracellular experiments however, it should be clear whether diamond particles influence cell biology. 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 concentrations 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 concentrations, the cellular metabolism 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 concentration and size to use for non-toxic, intracellular measurements in favour of (cancer) research in HeLa cells.

Generally Applicable Transformation Protocols for Fluorescent Nanodiamond Internalization into Cells.

Fluorescent nanodiamonds (FNDs) are promising nanoprobes, owing to their stable and magnetosensitive fluorescence. Therefore they can probe properties as magnetic resonances, pressure, temperature or strain. The unprecedented sensitivity of diamond defects can detect the faint magnetic resonance of a single electron or even a few nuclear spins. However, these sensitivities are only achieved if the diamond probe is close to the molecules that need to be detected. In order to utilize its full potential for biological applications, the diamond particle has to enter the cell. Some model systems, like HeLa cells, readily ingest particles. However, most cells do not show this behavior. In this article we show for the first time generally applicable methods, which are able to transport fluorescent nanodiamonds into cells with a thick cell wall. Yeast cells, in particular Saccharomyces cerevisiae, are a favored model organism to study intracellular processes including aging on a cellular level. In order to introduce FNDs in these cells, we evaluated electrical transformation and conditions of chemical permeabilization for uptake efficiency and viability. 5% DMSO (dimethyl sulfoxide) in combination with optimized chemical transformation mix leads to high uptake efficiency in combination with low impact on cell biology. We have evaluated all steps in the procedure.

The interaction of fluorescent nanodiamond probes with cellular media.

Fluorescent nanodiamonds (FNDs) are promising tools to image cells, bioanalytes and physical quantities such as temperature, pressure, and electric or magnetic fields with nanometer resolution. To exploit their potential for intracellular applications, the FNDs have to be brought into contact with cell culture media. The interactions between the medium and the diamonds crucially influence sensitivity as well as the ability to enter cells. The authors demonstrate that certain proteins and salts spontaneously adhere to the FNDs and may cause aggregation. This is a first investigation on the fundamental questions on how (a) FNDs interact with the medium, and (b) which proteins and salts are being attracted. A differentiation between strongly binding and weakly binding proteins is made. Not all proteins participate in the formation of FND aggregates. Surprisingly, some main components in the medium seem to play no role in aggregation. Simple strategies to prevent aggregation are discussed. These include adding the proteins, which are naturally present in the cell culture to the diamonds first and then inserting them in the full medium. Graphical abstractSchematic of the interaction of nanodiamonds with cell culture medium. Certain proteins and salts adhere to the diamond surface and lead to aggregation or to formation of a protein corona.

Improving surface and defect center chemistry of fluorescent nanodiamonds for imaging purposes--a review.

Diamonds are widely used for jewelry owing to their superior optical properties accounting for their fascinating beauty. Beyond the sparkle, diamond is highly investigated in materials science for its remarkable properties. Recently, fluorescent defects in diamond, particularly the negatively charged nitrogen-vacancy (NV(-)) center, have gained much attention: The NV(-) center emits stable, nonbleaching fluorescence, and thus could be utilized in biolabeling, as a light source, or as a Förster resonance energy transfer donor. Even more remarkable are its spin properties: with the fluorescence intensity of the NV(-) center reacting to the presence of small magnetic fields, it can be utilized as a sensor for magnetic fields as small as the field of a single electron spin. However, a reproducible defect and surface and defect chemistry are crucial to all applications. In this article we review methods for using nanodiamonds for different imaging purposes. The article covers (1) dispersion of particles, (2) surface cleaning, (3) particle size selection and reduction, (4) defect properties, and (5) functionalization and attachment to nanostructures, e.g., scanning probe microscopy tips.

Hot spot or not: a comparison of spatial statistical methods to predict prospective malaria infections.

BACKGROUND: Within affected communities, Plasmodium falciparum infections may be skewed in distribution such that single or small clusters of households consistently harbour a disproportionate number of infected individuals throughout the year. Identifying these hotspots of malaria transmission would permit targeting of interventions and a more rapid reduction in malaria burden across the whole community. This study set out to compare different statistical methods of hotspot detection (SaTScan, kernel smoothing, weighted local prevalence) using different indicators (PCR positivity, AMA-1 and MSP-1 antibodies) for prediction of infection the following year. METHODS: Two full surveys of four villages in Mwanza, Tanzania were completed over consecutive years, 2010-2011. In both surveys, infection was assessed using nested polymerase chain reaction (nPCR). In addition in 2010, serologic markers (AMA-1 and MSP-119 antibodies) of exposure were assessed. Baseline clustering of infection and serological markers were assessed using three geospatial methods: spatial scan statistics, kernel analysis and weighted local prevalence analysis. Methods were compared in their ability to predict infection in the second year of the study using random effects logistic regression models, and comparisons of the area under the receiver operating curve (AUC) for each model. Sensitivity analysis was conducted to explore the effect of varying radius size for the kernel and weighted local prevalence methods and maximum population size for the spatial scan statistic. RESULTS: Guided by AUC values, the kernel method and spatial scan statistics appeared to be more predictive of infection in the following year. Hotspots of PCR-detected infection and seropositivity to AMA-1 were predictive of subsequent infection. For the kernel method, a 1 km window was optimal. Similarly, allowing hotspots to contain up to 50% of the population was a better predictor of infection in the second year using spatial scan statistics than smaller maximum population sizes. CONCLUSIONS: Clusters of AMA-1 seroprevalence or parasite prevalence that are predictive of infection a year later can be identified using geospatial models. Kernel smoothing using a 1 km window and spatial scan statistics both provided accurate prediction of future infection.