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

A fluorescent nanodiamond foundation for quantum sensing in cells

Free radicals play a major role in the aging process as well as a most diseases. However, we barely know anything about them. These tiny molecules have an extremely short lifespan and are difficult to measure, while their role in health related processes is considerable. Fluorescent nanodiamonds are very small diamonds which can shed a light on this research question. These diamonds emit a constant light in a controlled setting. This is possible due to a small defect in the structure of the diamond, which makes it fluorescent. When free radicals are present, the light changes, which allows measurement of the radicals. During my PhD I have laid the basis for these biological measurements. Not all cells automatically take up diamonds or the diamonds tend to aggregate in cellular medium. By changing the solutions in which we administer the diamonds, we can prevent these obstacles. I have also performed a very detailed analysis of the cellular response on diamond uptake. Conveniently, the cells hardly show any response to the diamond uptake, an important result for our future measurements. In addition, I have developed new ways of targeting the diamonds to specific places in the cells, to obtain location specific information. Finally I have determined the subcellular location of the diamonds using a new technique, based on integrated electron microscopy. During my work I have laid the foundation for promising cellular research of ageing and disease using fluorescent nanodiamonds

pH Sensitive Dextran Coated Fluorescent Nanodiamonds as a Biomarker for HeLa Cells Endocytic Pathway and Increased Cellular Uptake

Fluorescent nanodiamonds are a useful for biosensing of intracellular signaling networks or environmental changes (such as temperature, 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 numbers 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 visualise this pathway. Additionally, 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

Fluorescent nanodiamonds as free radical sensors in live mammalian cells

Free radicals are atoms and molecules that contain at least one unpaired electron. For decades, they have attracted the attention of researchers, due to their role in numerous biological processes, both in health and disease. Due to their high reactivity, however, free radicals have always been difficult to detect with high sensitivity and resolution. Nanodiamonds – diamond nanoparticles – have been used as labels due to their exceptional biocompatibility and the extremely stable fluorescence of their color centers. This fluorescence can be modulated by external factors, such as magnetic fields. This property has led to the idea of using nanodiamonds to sense the magnetic fields generated by free radicals in biological samples. This thesis explores the applications of nanodiamond-based magnetometry for free radical detection in live mammalian cells and tackles some of the challenges of this approach

Lysine-functionalized nanodiamonds: synthesis, characterization and potential as gene delivery agents

Detonation nanodiamonds (NDs), due to their 4-5 nm primary particle size, stable inert core, reactive surface, ability to form hydrogel, are emerging as intracellular delivery vehicle for small and large molecules. Despite several favorable characteristics, the use of NDs in biological systems is impeded by their high aggregation propensity in polar liquid medium. To develop NDs as potential gene delivery vectors, pristine carboxylated NDs (pNDs) were functionalized with lysine through covalent conjugation. Raman and FTIR spectroscopic determinations confirmed the functionalization of NDs with lysine molecules, while thermogravimetric analysis estimated a surface loading of 1.7 mmol/g. Through lysine-functionalization, the dispersion stability of NDs in water increased considerably, showing a zeta potential of +49 mV. The average particle size of pNDs as measured by dynamic light scattering was substantially reduced from 1281 to 21 nm after lysine functionalization. Atomic force microscopy further substantiated the disaggregation of pNDs achieved through lysine functionalization. The lysine-functionalized NDs (fNDs) were able to electrostatically bind and block the migration of the nucleic acids at a weight ratio of 5:1 and 20:1 of fNDs:pDNA and fNDs:siRNA, respectively, with a shift in zeta potential from negative to positive value. The particle size of the complexes stabilized around 110 nm for fNDs-pDNA and less than 280 nm for fNDs-siRNA at the weight ratios of 50:1 fNDs:nucleic acid. While the Raman-fluorescence maps were equivocal with regards to the cellular association of NDs, backscattering maps clearly indicated the interaction of the fNDs with the cells. Cellular internalization of a few fNDs was suggested by laser confocal scanning microscopy. MTT assay demonstrated no significant in vitro cytotoxicity of pNDs and fNDs in the concentration range from 4 to 250 µg/mL. Flow cytometeric assessment of the gene expression (GFP intensity measurements) suggested that a strong binding of siRNA with fNDs might have prevented the release of nucleic acid into the cytoplasm of the cells. Overall, in this study, stable aqueous dispersion of NDs was generated using a mechanochemical approach feasible at a small laboratory scale, and early evidence was presented that the fNDs can be optimized for safe delivery of nucleic acids into mammalian cells

In vitro Interaction of Nanoparticles with Mitochondria for Surface Enhanced Raman Spectroscopy and Cell Imaging

Mitochondria are an attractive target for the design of cancer therapy. One of the mechanisms by which chemotherapeutics destroy cancer cells is by inducing apoptosis through extrinsic or intrinsic apoptotic pathways. Extrinsic pathways target cell surface receptors whilst intrinsic pathways target mitochondria. Several studies have shown cancer cell destruction through the extrinsic pathways, which target cancer-specific overexpressed growth factor receptors on the cell membrane. Although the mitochondria dependent apoptotic process is well understood, its application in cancer therapy is still not well developed. Therefore, to design an effective cancer therapy targeting mitochondria, a good understanding in mitochondria dependent apoptotic process is required. Recent developments in nanotechnology have enabled live cell investigations and non-destructive methods to obtain cellular information. The availability of such information would assist to design methods of targeted apoptosis induction. In view of this, I report on studies towards development of cancer therapy where nanoparticles (NPs) were targeted to human cell mitochondria for two purposes: (a) development of cell-imaging tools to investigate the fundamental cell biological pathways inside cells and (b) induction of apoptosis by targeting nanoparticles to mitochondria. Current medical and biological fluorescent imaging methods are mainly based on dye markers, which are limited in light emission per molecule, as well as photostability. Consequently, NPs are gaining prominence for molecular imaging because of their strong and stable fluorescence. Additionally, in order to get insight of mitochondrial molecular information, I investigated the use of optical properties of gold nanoparticles (Au NPs) for surface enhanced Raman spectroscopy (SERS). In this study, two types of Au NPs - nanospheres (Au NS) and nanorods (Au NR) were investigated. Results from this study showed the enhancement effect of Au NPs in Raman spectra of mitochondria, especially in the region from 1500 to 1600 cm-1. In this region, normal Raman spectra of mitochondria showed the presence of some understated Raman peaks probably due to the excitation wavelength dependence. Au NRs showed a larger enhancement effect than Au NS with respect to the penetration depth of the plasmonic nearfield enhancement effect. Although, the details of the enhancement mechanism are beyond the current studies, Au NPs could be enhancing vibrations of aromatic residues in proteins. This study therefore showed that Au NPs could enhance Raman spectra of mitochondria and in addition the shape of the nanoparticles had a significant effect on SERS spectra. In living cells, I investigated some transfection methods and targeting of NPs to mitochondria or cytosolic actin subunits. I tested the performance of three transfection reagents to deliver nanodiamonds (NDs) into living cells. Antibody functionalized NDs were targeted to mitochondria or cytosolic actin subunits. Three transfection reagents were used: cationic liposomes PULSin™, the cell penetrating peptide protamine, and oligosaccharide modified polypropylene imine (PPI) dendrimers. Fluorescence imaging results revealed that dendrimers were the most efficient in delivering ND conjugates to targeted organelles. Protamine-mediated transfections appeared to target ND conjugates to intended organelles, although there was a tendency of unfunctionalized NDs to be directed to the nucleus. PULSin™-mediated transfection formed ND aggregates regardless of the functionalization moiety. This reflected the unsuitability of the cationic liposome to mediate ND transfections. Further, I investigated the potential use of Au NPs for cell imaging and photothermal lysis of mitochondria inside cells. Just as above, I also tested the performance of the three-transfection reagents mentioned above on transfection capacity of Au NPs into living cells. Using transmission electron microscopy (TEM), oligosaccharide modified dendrimers showed the best transfection of functionalized Au NPs. Further experiments explored the use of the nearfield enhancement effect of Au NPs in combination with low-level laser irradiation (LLLI) to induce apoptosis in living cells. Analysis of the apoptotic process using cytochrome c release showed that Au NPs induced apoptosis most probably through mechanical disruption of the outer mitochondrial membrane. However, apoptosis was significantly accelerated in cells with mitochondrially targeted Au NRs than in cells without Au NRs. This study showed successful targeting of Au NPs to mitochondria in living cells, and demonstrated the potential of using Au NPs in combination with laser irradiation to induce the mitochondria dependent apoptotic pathway. In conclusion, the potential use of Au NPs in SERS of mitochondria and the application of NDs for cell imaging of intracellular organelles were demonstrated. Lastly, Au NPs were targeted to mitochondria in living cells and could induce apoptosis due to mechanical disruption of the outer mitochondrial membrane. Consequently, application of low-level laser irradiation to Au NP transfected cells accelerated the apoptotic process

In vitro Interaction of Nanoparticles with Mitochondria for Surface Enhanced Raman Spectroscopy and Cell Imaging

Mitochondria are an attractive target for the design of cancer therapy. One of the mechanisms by which chemotherapeutics destroy cancer cells is by inducing apoptosis through extrinsic or intrinsic apoptotic pathways. Extrinsic pathways target cell surface receptors whilst intrinsic pathways target mitochondria. Several studies have shown cancer cell destruction through the extrinsic pathways, which target cancer-specific overexpressed growth factor receptors on the cell membrane. Although the mitochondria dependent apoptotic process is well understood, its application in cancer therapy is still not well developed. Therefore, to design an effective cancer therapy targeting mitochondria, a good understanding in mitochondria dependent apoptotic process is required. Recent developments in nanotechnology have enabled live cell investigations and non-destructive methods to obtain cellular information. The availability of such information would assist to design methods of targeted apoptosis induction. In view of this, I report on studies towards development of cancer therapy where nanoparticles (NPs) were targeted to human cell mitochondria for two purposes: (a) development of cell-imaging tools to investigate the fundamental cell biological pathways inside cells and (b) induction of apoptosis by targeting nanoparticles to mitochondria. Current medical and biological fluorescent imaging methods are mainly based on dye markers, which are limited in light emission per molecule, as well as photostability. Consequently, NPs are gaining prominence for molecular imaging because of their strong and stable fluorescence. Additionally, in order to get insight of mitochondrial molecular information, I investigated the use of optical properties of gold nanoparticles (Au NPs) for surface enhanced Raman spectroscopy (SERS). In this study, two types of Au NPs - nanospheres (Au NS) and nanorods (Au NR) were investigated. Results from this study showed the enhancement effect of Au NPs in Raman spectra of mitochondria, especially in the region from 1500 to 1600 cm-1. In this region, normal Raman spectra of mitochondria showed the presence of some understated Raman peaks probably due to the excitation wavelength dependence. Au NRs showed a larger enhancement effect than Au NS with respect to the penetration depth of the plasmonic nearfield enhancement effect. Although, the details of the enhancement mechanism are beyond the current studies, Au NPs could be enhancing vibrations of aromatic residues in proteins. This study therefore showed that Au NPs could enhance Raman spectra of mitochondria and in addition the shape of the nanoparticles had a significant effect on SERS spectra. In living cells, I investigated some transfection methods and targeting of NPs to mitochondria or cytosolic actin subunits. I tested the performance of three transfection reagents to deliver nanodiamonds (NDs) into living cells. Antibody functionalized NDs were targeted to mitochondria or cytosolic actin subunits. Three transfection reagents were used: cationic liposomes PULSin™, the cell penetrating peptide protamine, and oligosaccharide modified polypropylene imine (PPI) dendrimers. Fluorescence imaging results revealed that dendrimers were the most efficient in delivering ND conjugates to targeted organelles. Protamine-mediated transfections appeared to target ND conjugates to intended organelles, although there was a tendency of unfunctionalized NDs to be directed to the nucleus. PULSin™-mediated transfection formed ND aggregates regardless of the functionalization moiety. This reflected the unsuitability of the cationic liposome to mediate ND transfections. Further, I investigated the potential use of Au NPs for cell imaging and photothermal lysis of mitochondria inside cells. Just as above, I also tested the performance of the three-transfection reagents mentioned above on transfection capacity of Au NPs into living cells. Using transmission electron microscopy (TEM), oligosaccharide modified dendrimers showed the best transfection of functionalized Au NPs. Further experiments explored the use of the nearfield enhancement effect of Au NPs in combination with low-level laser irradiation (LLLI) to induce apoptosis in living cells. Analysis of the apoptotic process using cytochrome c release showed that Au NPs induced apoptosis most probably through mechanical disruption of the outer mitochondrial membrane. However, apoptosis was significantly accelerated in cells with mitochondrially targeted Au NRs than in cells without Au NRs. This study showed successful targeting of Au NPs to mitochondria in living cells, and demonstrated the potential of using Au NPs in combination with laser irradiation to induce the mitochondria dependent apoptotic pathway. In conclusion, the potential use of Au NPs in SERS of mitochondria and the application of NDs for cell imaging of intracellular organelles were demonstrated. Lastly, Au NPs were targeted to mitochondria in living cells and could induce apoptosis due to mechanical disruption of the outer mitochondrial membrane. Consequently, application of low-level laser irradiation to Au NP transfected cells accelerated the apoptotic process

Targeted Nanodiamonds for Identification of Subcellular Protein Assemblies in Mammalian Cells

Transmission electron microscopy (TEM) can be used to successfully determine the structures of proteins. However, such studies are typically done ex situ after extraction of the protein from the cellular environment. Here we describe an application for nanodiamonds as targeted intensity contrast labels in biological TEM, using the nuclear pore complex (NPC) as a model macroassembly. We demonstrate that delivery of antibody-conjugated nanodiamonds to live mammalian cells using maltotriose-conjugated polypropylenimine dendrimers results in efficient localization of nanodiamonds to the intended cellular target. We further identify signatures of nanodiamonds under TEM that allow for unambiguous identification of individual nanodiamonds from a resin-embedded, OsO4-stained environment. This is the first demonstration of nanodiamonds as labels for nanoscale TEM-based identification of subcellular protein assemblies. These results, combined with the unique fluorescence properties and biocompatibility of nanodiamonds, represent an important step toward the use of nanodiamonds as markers for correlated optical/electron bioimaging.Comment: 38 pages, 6 figures, SI section with 3 figure