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.

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.

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.

X-ray fluorescence computed tomography (XFCT) is an emerging biomedical imaging technique. The KTH-XFCT laboratory system offers a characteristic 24 keV emission line, and high spatial resolution (200 𝜇m) images. XFCT as a newly emerging modality also requires the exploration and development of suitable contrast agents. Nanomaterials have been widely used as contrast agents in many popular imaging modalities like MRI, PET, and CT. They have several advantages including long blood circulation time, high ratio of surface area to volume, and enhanced image contrast. However, the use of nanomaterials as contrast agents is limited by their biocompatibility and toxicity, which are determined by the physicochemical properties including size, morphology, surface chemistry. Therefore, the study on the synthesis and characterization of nanomaterials is an indispensable step. In this thesis, a group of elements (Y, Zr, Nb, Ru, Rh) are selected based on the X-ray K𝛼-absorption energy, matching with the 24 keV emission line of KTH-XFCT source. Y, Zr, Nb, Ru and Rh based nanoparticles are synthesized by hydrothermal and polyol method, identified as the ceramic and metallic groups. XRF performance is demonstrated by the XFCT system. Metallic Ru and Rh nanoparticles are further selected to study the synthesis conditions and in vitro toxicity for their smaller TEM and hydrodynamic size. Surface properties are investigated to show the isoelectric point and polymer coating on the metallic nanoparticles. Morphological different Rh nanoparticles are obtained by introducing different additives during the synthesis, indicating the different cytotoxicity performance attributed to different morphologies. Silica coating is further performed on the surface of metallic and metallic nanoparticles to improve their biocompatibility. The in vitro toxicity assessment are performed on the murine macrophages and human ovarian cancer cell lines. X-ray fluorescence performance is evaluated for each nanoparticles by using soft-tissue equivalent holder and in situ small-animal imaging experiments. The results indicates the spatial resolution and detection sensitivity of the concentration of the metallic nanoparticles. In this work, we demonstrate the potential of a group selected nanomaterials as XFCT contrast agents for the first time, especially, investigate the synthesis, surface properties, in vitro toxicity as well as detection sensitivity of the metallic nanoparticles.

Röntgenfluorescenstomografi (XFCT) är en ny och lovande teknik för biomedicinsk avbildning. Det kompakta KTH-XFCT systemet har en röntgenemission på 24 keV och möjliggör avbildning med hög spatial upplösning (200 𝜇m). Eftersom XFCT är en ny modalitet behövs forskning och utveckling av lämpliga kontrastmedel. Nanomaterial har använts brett som kontrastmedel inom många etablerade avbildningstekniker, så som magnetresonanstomografi (MR), positronemissionstomografi (PET) och datortomografi (CT). Nanomaterial har flera fördelar jämfört med traditionella kontrastmedel, exempelvis längre blodcirkulationstider, större yta per volym och överlag förbättrad kontrast. Däremot begränsas användningen av nanomaterial som kontrastmedel av egenskaper såsom biokompatibilitet och toxicitet vilket bestäms av fysikokemikaliska egenskaper såsom storlek, morfologi och ytkemi. Därför är studier av syntes samt karakterisering av nanomaterial viktigt för biomedicinska tillämpningar. I denna avhandling har en grupp grundämnen (Y, Zr, Nb, Ru, Rh) valts ut baserat på att deras K-absorptionskanter matchar röntgenemissionen på 24 keV hos KTH-XFCT systemet. Y, Zr, Nb, Ru och Rh nanopartiklar synteserades med hydrotermiska samt polyol-metoder och klassades som antingen keramiska eller metalliska nanopartiklar. Röntgenfluorescensen från nanopartiklarna påvisades med KTH-XFCT systemet. De metalliska nanopartiklarna med mindre storlek baserade på Ru och Rh valdes ut och studerades närmare i termer av syntesparametrar samt in vitro toxicitet i cellkulturer. Ytegenskaper hos dessa metalliska nanopartiklar undersöktes för att uppvisa deras isoelektriska punkter samt PVP-ytskikt. Morfologiskt kontrollerbara Rh nanopartiklar tillverkades genom att introducera olika tillsatser under syntesprocessen, vilket sedan användes för att påvisa att cytotoxiciteten är morfologi-beroende. Ytan hos de metalliska nanopartiklarna modifierades med ett skikt av kiseldioxid för att ytterligare förbättra biokompatibiliteten. Murina makrofager och mänskliga cellinjer för äggstockscancer användes för att evaluera toxiciteten in vitro. Röntgenfluorescensavbildning med nanopartiklarna demonstrerades både med hjälp av mjukvävnad-liknande fantomer samt med in situ experiment i avlivna möss. Resultaten indikerade både spatial upplösning och observerbar koncentration av ackumulerade nanopartiklar i KTH-XFCT systemet. I detta arbete demonstreras för första gången potentialen hos en grupp utvalda nanomaterial som kontrastmedel för XFCT. Detta arbete inkluderar studier av syntes, ytkemi, in vitro toxicitet samt detektionssensitiviteten hos de metalliska nanopartiklarna.

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. 

Nanoparticles (NPs) have been used as contrast agents for several bioimaging modalities. X-ray fluorescence (XRF) tomography can provide sensitive and quantitative 3D detection of NPs. With spectrally matched NPs as contrast agents, we demonstrated earlier in a laboratory system that XRF tomography could achieve high-spatial-resolution tumor imaging in mice. Here, we present the synthesis, characterization, and evaluation of a library of NPs containing Y, Zr, Nb, Rh, and Ru that have spectrally matched K-shell absorption for the laboratory scale X-ray source. The K-shell emissions of these NPs are spectrally well separated from the X-ray probe and the Compton background, making them suitable for the lab-scale XRF tomography system. Their potential as XRF contrast agents is demonstrated successfully in a small-animal equivalent phantom, confirming the simulation results. The diversity in the NP composition provides a flexible platform for a better design and biological optimization of XRF tomography nanoprobes.