Arthur Taylor

Photothermal microscopy of the core of dextran-coated iron oxide nanoparticles during cell uptake.

A detailed understanding of cellular interactions with superparamagnetic iron oxide nanoparticles (SPIONs) is critical when their biomedical applications are considered. We demonstrate how photothermal microscopy can be used to follow the cellular uptake of SPIONs by direct imaging of the iron oxide core. This offers two important advantages when compared with current strategies employed to image magnetic cores: first, it is nondestructive and is therefore suitable for studies of live cells and, second, it offers a higher sensitivity and resolution, thus allowing for the identification of low levels of SPIONs within a precise subcellular location. We have shown that this technique may be applied to the imaging of both cell monolayers and cryosections. In the former we have demonstrated the role of temperature on the rate of endocytosis, while in the latter we have been able to identify cells labeled with SPIONs from a mixed population containing predominantly unlabeled cells. Direct imaging of the SPION core is of particular relevance for research involving clinically approved SPIONs, which do not contain fluorescent tags and therefore cannot be detected via fluorescence microscopy.

A carbon-wrapped nanoscaled thermometer for temperature control in biological environments

AIMS A carbon-wrapped nanoscaled thermometer for a contactless temperature control in biological systems on the cellular level is presented. MATERIALS & METHODS The thermometer is based on multiwalled carbon nanotubes (MWCNTs) filled with materials with strongly temperature-dependent nuclear magnetic resonance (NMR) parameters. The NMR frequency shift and relaxation time were measured in cuprous-iodide-filled CNTs at different temperatures. RESULTS The experimental data indicate a pronounced temperature dependence of the NMR parameters, thereby realizing the nanoscaled thermometer. CONCLUSION This study is a proof-of-concept that the functionalized CNTs can be used as a contactless thermometer in biomedical applications.

Assessing the Efficacy of Nano- and Micro-Sized Magnetic Particles as Contrast Agents for MRI Cell Tracking

Iron-oxide based contrast agents play an important role in magnetic resonance imaging (MRI) of labelled cells in vivo. Currently, a wide range of such contrast agents is available with sizes varying from several nanometers up to a few micrometers and consisting of single or multiple magnetic cores. Here, we evaluate the effectiveness of these different particles for labelling and imaging stem cells, using a mouse mesenchymal stem cell line to investigate intracellular uptake, retention and processing of nano- and microsized contrast agents. The effect of intracellular confinement on transverse relaxivity was measured by MRI at 7 T and in compliance with the principles of the ‘3Rs’, the suitability of the contrast agents for MR-based cell tracking in vivo was tested using a chick embryo model. We show that for all particles tested, relaxivity was markedly reduced following cellular internalisation, indicating that contrast agent relaxivity in colloidal suspension does not accurately predict performance in MR-based cell tracking studies. Using a bimodal imaging approach comprising fluorescence and MRI, we demonstrate that labelled MSC remain viable following in vivo transplantation and can be tracked effectively using MRI. Importantly, our data suggest that larger particles might confer advantages for longer-term imaging.

Overexpression of the MRI Reporter Genes Ferritin and Transferrin Receptor Affect Iron Homeostasis and Produce Limited Contrast in Mesenchymal Stem Cells

Imaging technologies that allow the non-invasive monitoring of stem cells in vivo play a vital role in cell-based regenerative therapies. Recently, much interest has been generated in reporter genes that enable simultaneous monitoring of the anatomical location and viability of cells using magnetic resonance imaging (MRI). Here, we investigate the efficacy of ferritin heavy chain-1 (Fth1) and transferrin receptor-1 (TfR1) as reporters for tracking mesenchymal stem cells. The overexpression of TfR1 was well tolerated by the cells but Fth1 was found to affect the cell’s iron homeostasis, leading to phenotypic changes in the absence of iron supplementation and an upregulation in transcript and protein levels of the cell’s endogenous transferrin receptor. Neither the sole overexpression of Fth1 nor TfR1 resulted in significant increases in intracellular iron content, although significant differences were seen when the two reporter genes were used in combination, in the presence of high concentrations of iron. The supplementation of the culture medium with iron sources was a more efficient means to obtain contrast than the use of reporter genes, where high levels of intracellular iron were reflected in transverse (T2) relaxation. The feasibility of imaging iron-supplemented cells by MRI is shown using a 3R-compliant chick embryo model.

Influence of Nd:YAG laser irradiation on an adhesive restorative procedure.

Hard tissue modification by means of laser irradiation is becoming popular in dentistry, since it promotes assorted responses between the tooth and the restorative material. Some studies on the bond strength of adhesive systems to Nd:YAG irradiated teeth have shown distinctive behaviors when irradiation was applied before or after the adhesive agent. This study evaluated the microtensile bond strength of a commercial adhesive system to dentin irradiated with Nd:YAG laser after adhesive application but prior to polymerization. The experiment was conducted in vitro, using freshly extracted human teeth as samples. For the microtensile test, the teeth were separated into 4 different groups according to the energy density of laser irradiation: 0, 5, 10 and 50 J/cm2. The data was analyzed with analysis of variance (ANOVA) and LSD tests, and the results indicated that the group that was irradiated with 5 J/cm2 had significantly higher bond strength values. Adhesive penetration on the etched dentin was observed by scanning electron microscopy, where the images showed better adhesive penetration on dentinal tubules after dentin irradiation with 5 J/cm2. Based on the results of this study, it is possible to conclude that irradiation of dentin with the Nd:YAG laser at low energy densities after application of the adhesive but prior to polymerization might be positive for the adhesive restorative process.

Preventing Plasmon Coupling between Gold Nanorods Improves the Sensitivity of Photoacoustic Detection of Labeled Stem Cells in Vivo.

Gold nanorods are excellent contrast agents for imaging technologies which rely on near-infrared absorption such as photoacoustic imaging. For cell tracking applications, the cells of interest are labeled with the contrast agent prior to injection. However, after uptake into cells by endocytosis, the confinement and high concentration in endosomes leads to plasmon band broadening and reduced absorbance. This would limit the potential of multispectral optoacoustic tomography in terms of spectral processing and, consequently, sensitivity. Here, we show that steric hindrance provided by silica coating of the nanorods leads to the preservation of their spectral properties and improved photoacoustic sensitivity. This strategy allowed the detection and monitoring of as few as 2 × 10(4) mesenchymal stem cells in mice over a period of 15 days with a high spatial resolution. Importantly, the silica-coated nanorods did not affect the viability or differentiation potential of the transplanted mesenchymal stem cells.

Evaluating the effectiveness of transferrin receptor-1 (TfR1) as a magnetic resonance reporter gene.

Magnetic resonance (MR) reporter genes have the potential for tracking the biodistribution and fate of cells in vivo, thus allowing the safety, efficacy and mechanisms of action of cell‐based therapies to be comprehensively assessed. In this study, we evaluate the effectiveness of the iron importer transferrin receptor‐1 (TfR1) as an MR reporter gene in the model cell line CHO‐K1. Overexpression of the TfR1 transgene led to a reduction in the levels of endogenous TfR1 mRNA, but to a 60‐fold increase in total TfR1 protein levels. Although the mRNA levels of ferritin heavy chain‐1 (Fth1) did not change, Fth1 protein levels increased 13‐fold. The concentration of intracellular iron increased significantly, even when cells were cultured in medium that was not supplemented with iron and the amount of iron in the extracellular environment was thus at physiological levels. However, we found that, by supplementing the cell culture medium with ferric citrate, a comparable degree of iron uptake and MR contrast could be achieved in control cells that did not express the TfR1 transgene. Sufficient MR contrast to enable the cells to be detected in vivo following their administration into the midbrain of chick embryos was obtained irrespective of the reporter gene. We conclude that TfR1 is not an effective reporter and that, to track the biodistribution of cells with MR imaging in the short term, it is sufficient to simply culture cells in the presence of ferric citrate. Copyright

Imaging technologies for monitoring the safety, efficacy and mechanisms of action of cell-based regenerative medicine therapies in models of kidney disease

The incidence of end stage kidney disease is rising annually and it is now a global public health problem. Current treatment options are dialysis or renal transplantation, which apart from their significant drawbacks in terms of increased morbidity and mortality, are placing an increasing economic burden on society. Cell-based Regenerative Medicine Therapies (RMTs) have shown great promise in rodent models of kidney disease, but clinical translation is hampered due to the lack of adequate safety and efficacy data. Furthermore, the mechanisms whereby the cell-based RMTs ameliorate injury are ill-defined. For instance, it is not always clear if the cells directly replace damaged renal tissue, or whether paracrine effects are more important. Knowledge of the mechanisms responsible for the beneficial effects of cell therapies is crucial because it could lead to the development of safer and more effective RMTs in the future. To address these questions, novel in vivo imaging strategies are needed to monitor the biodistribution of cell-based RMTs and evaluate their beneficial effects on host tissues and organs, as well as any potential adverse effects. In this review we will discuss how state-of-the-art imaging modalities, including bioluminescence, magnetic resonance, nuclear imaging, ultrasound and an emerging imaging technology called multispectral optoacoustic tomography, can be used in combination with various imaging probes to track the fate and biodistribution of cell-based RMTs in rodent models of kidney disease, and evaluate their effect on renal function.

Development of Multifunctional Magnetic Nanoparticles for Genetic Engineering and Tracking of Neural Stem Cells

Genetic modification of cell transplant populations and cell tracking ability are key underpinnings for effective cell therapies. Current strategies to achieve these goals utilize methods which are unsuitable for clinical translation because of related safety issues, and multiple protocol steps adding to cost and complexity. Multifunctional magnetic nanoparticles (MNPs) offering dual mode gene delivery and imaging contrast capacity offer a valuable tool in this context. Despite their key benefits, there is a critical lack of neurocompatible and multifunctional particles described for use with transplant populations for neurological applications. Here, a systematic screen of MNPs (using a core shown to cause contrast in magnetic resonance imaging (MRI)) bearing various surface chemistries (polyethylenimine (PEI) and oxidized PEI and hybrids of oxidized PEI/alginic acid, PEI/chitosan and PEI/polyamidoamine) is performed to test their ability to genetically engineer neural stem cells (NSCs; a cell population of high clinical relevance for central nervous system disorders). It is demonstrated that gene delivery to NSCs can be safely achieved using two of the developed formulations (PEI and oxPEI/alginic acid) when used in conjunction with oscillating magnetofection technology. After transfection, intracellular particles can be detected by histological procedures with labeled cells displaying contrast in MRI (for real time cell tracking).

Co-precipitation of DEAE-dextran coated SPIONs: how synthesis conditions affect particle properties, stem cell labelling and MR contrast†

Superparamagnetic iron oxide nanoparticles (SPIONs) are widely used as contrast agents for stem cell tracking using magnetic resonance imaging (MRI). The total mass of iron oxide that can be internalised into cells without altering their viability or phenotype is an important criterion for the generation of contrast, with SPIONs designed for efficient labelling of stem cells allowing for an increased sensitivity of detection. Although changes in the ratio of polymer and iron salts in co-precipitation reactions are known to affect the physicochemical properties of SPIONs, particularly core size, the effects of these synthesis conditions on stem cell labelling and magnetic resonance (MR) contrast have not been established. Here, we synthesised a series of cationic SPIONs with very similar hydrodynamic diameters and surface charges, but different polymer content. We have investigated how the amount of polymer in the co-precipitation reaction affects core size and modulates not only the magnetic properties of the SPIONs but also their uptake into stem cells. SPIONs with the largest core size and lowest polymer content presented the highest magnetisation and relaxivity. These particles also had the greatest uptake efficiency without any deleterious effect on either the viability or function of the stem cells. However, for all particles internalised in cells, the T2 and T2* relaxivity was independent of the SPIONs core size. Our results indicate that the relative mass of iron taken up by cells is the major determinant of MR contrast generation and suggest that the extent of SPION uptake can be regulated by the amount of polymer used in co-precipitation reactions. Copyright