Aims: To investigate the distribution and toxicity of ruthenium nanoparticles (Ru NPs) injected intravenously in mice.

Methods: We synthesized Ru NPs, followed their biodistribution by x-ray fluorescence (XRF) imaging and evaluated organ toxicity by histopathology and gene expression.

Results: Ru NPs accumulated, mainly in liver and spleen, where they were phagocyted by tissue macrophages, giving a transient inflammation and oxidative stress response that declined after 2 weeks. Ru NPs gradually accumulated in the skin, which was confirmed by microscopic examination of skin biopsies.

Conclusion: Ru NP toxicity in recipient organs is transient. Particles are at least partially excreted by the skin, supporting a role for the skin as a nanoparticle clearing organ.

In recent years, the design and synthesis of bio-compatible coatings leading to hybrid nanoparticles (NPs) as the contrast agents have gained substantial relevance. Furthermore, the addition of several functionalities for bio-imaging applications represents a key step for non-invasive bio-diagnostics. In this context, we design and utilize hybrid nanostructures for X-ray fluorescence computed tomography (XFCT). The combination of a ceramic or metallic core–based on MoO2, Rh or Ru–with a protective shell allows the generation of bio-compatible nanohybrids for dual mode bio-imaging, where the core NPs constitute the X-ray fluorescence (XRF) contrast agents [1]–[3]. Core NPs are synthesized via polyol, hydrothermal or microwave-assisted hydrothermal methods, yielding uniform shape and high dispersibility in aqueous media. Different approaches have been pursued for the fabrication of a bio-compatible shell coating. A modified sol-gel based silica coating process, doped with a commercial fluorophore (Cy5.5), was developed and shown to be applicable to both ceramic and metallic NPs [4], forming core-shell NPs with both optical and X-ray fluorescence properties. Alternatively, carbon quantum dots (CQDs) were synthesized via citrate pyrolysis using microwave-assisted hydrothermal method, exhibiting uniform size distribution (1.6±0.4 nm) and excitation-independent emission (440 nm). Conjugation of these CQDs, via cross-linking, with Rh NPs led to excitation-independent hybrid NPs, with a red-shifted emission wavelength (520 nm), attributed to the reduction of pyrrolic nitrogen on CQDs [5]. These hybrid NPs exhibit improved in vitro biocompatibility in comparison with bare XRF contrast agents. Furthermore, the optical fluorescence–provided by Cy5.5 or CQDs–allows the localization of the NPs in the intracellular environment while the XRF signal from the core NPs is utilized for XFCT, in small animals, leading to both a microscopic and macroscopic bio-imaging contrast agent.

Multimodal contrast agents in biomedical imaging enable the collection of more comprehensive diagnostic information. In the present work, we design hybrid ruthenium-decorated superparamagnetic iron oxide nanoparticles (NPs) as the contrast agents for both magnetic resonance imaging (MRI) and X-ray fluorescence computed tomography (XFCT). The NPs are synthesized via a one-pot polyol hot injection route, in diethylene glycol. In vivo preclinical studies demonstrate the possibility of correlative bioimaging with these contrast agents. The complementarity allows accurate localization, provided by the high contrast of the soft tissues in MRI combined with the elemental selectivity of XFCT, leading to NP detection with high specificity and resolution. We envision that this multimodal imaging could find future applications for early tumor diagnosis, improved long-term treatment monitoring, and enhanced radiotherapy planning.

Nanoparticle (NP) based contrast agents detectable via different imaging modalities (multimodal properties) provide a promising strategy for noninvasive diagnostics. Core-shell NPs combining optical and X-ray fluorescence properties as bioimaging contrast agents are presented. NPs developed earlier for X-ray fluorescence computed tomography (XFCT), based on ceramic molybdenum oxide (MoO2) and metallic rhodium (Rh) and ruthenium (Ru), are coated with a silica (SiO2) shell, using ethanolamine as the catalyst. The SiO2 coating method introduced here is demonstrated to be applicable to both metallic and ceramic NPs. Furthermore, a fluorophore (Cy5.5 dye) was conjugated to the SiO2 layer, without altering the morphological and size characteristics of the hybrid NPs, rendering them with optical fluorescence properties. The improved biocompatibility of the SiO2 coated NPs without and with Cy5.5 is demonstrated in vitro by Real-Time Cell Analysis (RTCA) on a macrophage cell line (RAW 264.7). The multimodal characteristics of the core-shell NPs are confirmed with confocal microscopy, allowing the intracellular localization of these NPs in vitro to be tracked and studied. In situ XFCT successfully showed the possibility of in vivo multiplexed bioimaging for multitargeting studies with minimum radiation dose. Combined optical and X-ray fluorescence properties empower these NPs as effective macroscopic and microscopic imaging tools.

Surface derivatized magnetic nanoparticles have been commonly used for magnetic separation. Facile mechanisms are needed to be developed for the design of bio-functionalized magnetic hybrid materials, where the surfaces can be re-generated for the re-use of the developed platforms. Superparamagnetic iron oxide nanoparticles with a diameter below 10 nm were synthesized via a novel microwave-assisted hydrothermal method in the presence of citrate ions, which allowed to obtain uniform and negatively charged nanoparticles. These were then coupled with Poly-l-lysine (PLL), forming micrometer-sized self-assembled spherical entities. Cross-linking the PLL within these microspheres with glutaraldehyde stabilized them chemically and mechanically. The active bio-functionality was introduced by a protein grafting methodology, using m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (SMBS). The Moringa oleifera Coagulant Protein (MOCP) from a seed extract was employed for its characteristic coagulation activity. The performance of the MOCP functionalized microspheres was evaluated as a function of turbidity removal of problematic colloidal clay from water via magnetic separation, resulting in over 80% of activity within 15 min. Surface of these hybrid materials can be re-generated by treatment with alcohol, allowing their easy magnetic separation and re-use. The rapid and strong response with tunable magnetic property makes these hybrid microspheres a powerful tool for many potential applications, due to the general applicability of the developed methodology.

X-ray fluorescence computed tomography (XFCT) with nanoparticles (NPs) as contrast agents shows potential for molecular biomedical imaging with higher spatial resolution than present methods. To date the technique has been demonstrated on phantoms and mice, however, parameters such as radiation dose, exposure times and sensitivity have not yet allowed for high-spatial-resolution in vivo longitudinal imaging, i.e., imaging of the same animal at different time points. Here we show in vivo XFCT with spatial resolution in the 200-400 mu m range in a proof-of-principle longitudinal study where mice are imaged five times each during an eight-week period following tail-vein injection of NPs. We rely on a 24 keV x-ray pencil-beam-based excitation of in-house-synthesized molybdenum oxide NPs (MoO2) to provide the high signal-to-background x-ray fluorescence detection necessary for XFCT imaging with low radiation dose and short exposure times. We quantify the uptake and clearance of NPs in vivo through imaging, and monitor animal well-being over the course of the study with support from histology and DNA stability analysis to assess the impact of x-ray exposure and NPs on animal welfare. We conclude that the presented imaging arrangement has potential for in vivo longitudinal studies, putting emphasis on designing biocompatible NPs as the future focus for active-targeting preclinical XFCT.

Understanding the biological fate of graphene-based materials such as graphene oxide (GO) is crucial to assess adverse effects following intentional or inadvertent exposure. Here we provide first evidence of biodegradation of GO in the gastrointestinal tract using zebrafish as a model. Raman mapping was deployed to assess biodegradation. The degradation was blocked upon knockdown ofnos2aencoding the inducible nitric oxide synthase (iNOS) or by pharmacological inhibition of NOS usingl-NAME, demonstrating that the process was nitric oxide (NO)-dependent. NO-dependent degradation of GO was further confirmedin vitroby combining a superoxide-generating system, xanthine/xanthine oxidase (X/XO), with an NO donor (PAPA NONOate), or by simultaneously producing superoxide and NO by decomposition of SIN-1. Finally, by using the transgenic strainTg(mpx:eGFP) to visualize the movement of neutrophils, we could show that inhibition of the degradation of GO resulted in increased neutrophil infiltration into the gastrointestinal tract, indicative of inflammation.

X-Ray fluorescence computed tomography (XFCT) is an emerging biomedical imaging technique, which demands the development of new contrast agents. Ruthenium (Ru) and rhodium (Rh) have spectrally attractive K ff edge energies, qualifying them as new XFCT bio-imaging probes. Metallic Ru and Rh nanoparticles are synthesized by polyol method, in the presence of a stabilizer. The effect of several reaction parameters, including reaction temperature time, precursor and stabilizer concentration, and stabilizer molecular weight, on the size of particles, were studied. Resultant materials were characterized in detail using XRD, TEM, FT-IR, DLS-zeta potential and TGA techniques. Ru particles in the size range of 1-3 nm, and Rh particles of 6-9 nm were obtained. At physiological pH, both material systems showed agglomeration into larger assemblies ranging from 12-104 nm for Ru and 25-50 nm for Rh. Cytotoxicity of the nanoparticles (NPs) was evaluated on macrophages and ovarian cancer cells, showing minimal toxicity in doses up to 50 mu g/mL. XFCT performance was evaluated on a small-animal-sized phantom model, demonstrating the possibility of quantitative evaluation of the measured dose with an expected linear response. This work provides a detailed route for the synthesis, size control and characterization of two materials systems as viable contrast agents for XFCT bio-imaging.

Morphologically controllable synthesis of Rh nanoparticles (NPs) was achieved by the use of additives during polyol synthesis. The effect of salts and surfactant additives including PVP, sodium acetate, sodiumcitrate, CTAB,CTAC,andpotassiumbromideonRhNPsmorphologywasinvestigated. When PVP was used as the only additive, trigonal NPs were obtained. Additives containing Br− ions (CTAB and KBr) resulted in NPs with a cubic morphology, while those with carboxyl groups (sodium citrate and acetate) formed spheroid NPs. The use of Cl− ions (CTAC) resulted in a mixture of polygon morphologies. Cytotoxicity of these NPs was evaluated on macrophages and ovarian cancer cell lines. Membrane integrity and cellular activity are both influenced to a similar extent, for both the cell lines, with respect to the morphology of Rh NPs. The cells exposed to trigonal Rh NPs showed the highest viability, among the NP series. Particles with a mixed polygon morphology had the highest cytotoxic impact, followed by cubic and spherical NPs. The Rh NPs were further demonstrated as contrast agents for X-ray fluorescence computed tomography (XFCT) in a small-animal imaging setting. This work provides a detailed route for the synthesis, morphology control, and characterization of Rh NPs as viable contrast agents for XFCT bio-imaging. 

Background Colistin is a polypeptide antibiotic drug that targets lipopolysaccharides in the outer membrane of Gram-negative bacteria. Inactivation of the mgrB-gene is a common mechanism behind colistin-resistance in Klebsiella pneumoniae (Kpn). Since colistin is a cyclic polypeptide, it may exhibit cross-resistance with the antimicrobial peptide LL-37, and with other innate effector mechanisms, but previous results are inconclusive. Objective To study potential cross-resistance between colistin and LL-37, as well as with other innate effector mechanisms, and to compare virulence of colistin-resistant and susceptible Kpn strains. Materials/Methods Carbapenemase-producing Kpn from Oman (n = 17) were subjected to antimicrobial susceptibility testing and whole genome sequencing. Susceptibility to colistin and LL-37 was studied. The surface charge was determined by zeta-potential measurements and the morphology of treated bacteria was analyzed with electron microscopy. Bacterial survival was assessed in human whole blood and serum, as well as in a zebrafish infection-model. Results Genome-analysis revealed insertion-sequences in the mgrB gene, as a cause of colistin resistance in 8/17 isolates. Colistin-resistant (Col-R) isolates were found to be more resistant to LL-37 compared to colistin-susceptible (Col-S) isolates, but only at concentrations >= 50 mu g/ml. There was no significant difference in surface charge between the isolates. The morphological changes were similar in both Col-R and Col-S isolates after exposure to LL-37. Finally, no survival difference between the Col-R and Col-S isolates was observed in whole blood or serum, or in zebrafish embryos. Conclusion Cross-resistance between colistin and LL-37 was observed at elevated concentrations of LL-37. However, Col-R and Col-S isolates exhibited similar survival in serum and whole blood, and in a zebrafish infection-model, suggesting that cross-resistance most likely play a limited role during physiological conditions. However, it cannot be ruled out that the observed cross-resistance could be relevant in conditions where LL-37 levels reach high concentrations, such as during infection or inflammation.