Synovial sarcoma (SS) is driven by a unique t(18;X) chromosomal translocation resulting in expression of the SS18-SSX fusion oncoprotein, a transcriptional regulator with both activating and repressing functions. However, the manner in which SS18-SSX contributes to the development of SS is not entirely known. Here, we show that SS18-SSX drives the expression of Preferentially Expressed Antigen in Melanoma (PRAME), which is highly expressed in SS but whose function remains poorly understood. The fusion protein directly binds and activates the PRAME promoter and we found that expression of SS18-SSX and PRAME are positively correlated. We provide evidence that PRAME modulates retinoic acid (RA) signaling, forming a ternary complex with the RA receptor α (RARα) and the Enhancer of Zeste Homolog 2 (EZH2). Knockdown of PRAME suppressed the response to all-trans retinoic acid (ATRA) supporting PRAME’s role in modulating RA-signaling. Notably, we demonstrate that combined pharmacological inhibition of EZH2 and treatment with ATRA reconstituted RA signaling followed by reduced proliferation and induction of cellular senescence. In conclusion, our data provides new insights on the role of the SS18-SSX fusion protein in regulation of PRAME expression and RA signaling, highlighting the therapeutic potential of disrupting the RARα-PRAME-EZH2 complex in SS. (Figure presented.)

Nanoparticles (NPs) are currently developed for drug delivery and molecular imaging. However, they often get intercepted before reaching their target, leading to low targeting efficacy and signal-to-noise ratio. They tend to accumulate in organs like lungs, liver, kidneys, and spleen. The remedy is to iteratively engineer NP surface properties and administration strategies, presently a time-consuming process that includes organ dissection at different time points. To improve this, we propose a rapid iterative approach using whole-animal x-ray fluorescence (XRF) imaging to systematically evaluate NP distribution in vivo. We applied this method to molybdenum-based NPs and clodronate liposomes for tumor targeting with transient macrophage depletion, leading to reduced accumulations in lungs and liver and eventual tumor detection. XRF computed tomography (XFCT) provided 3D insight into NP distribution within the tumor. We validated the results using a multiscale imaging approach with dye-doped NPs and gene expression analysis for nanotoxicological profiling. XRF imaging holds potential for advancing therapeutics and diagnostics in preclinical pharmacokinetic studies.

Diffraction-limited resolution and low penetration depth are fundamental constraints in optical microscopy and in vivo imaging. Recently, liquid-jet X-ray technology has enabled the generation of X-rays with high-power intensities in laboratory settings. By allowing the observation of cellular processes in their natural state, liquid-jet soft X-ray microscopy (SXM) can provide morphological information on living cells without staining. Furthermore, X-ray fluorescence imaging (XFI) permits the tracking of contrast agents in vivo with high elemental specificity, going beyond attenuation contrast. In this study, we established a methodology to investigate nanoparticle (NP) interactions in vitro and in vivo, solely based on X-ray imaging. We employed soft (0.5 keV) and hard (24 keV) X-rays for cellular studies and preclinical evaluations, respectively. Our results demonstrated the possibility of localizing NPs in the intracellular environment via SXM and evaluating their biodistribution with in vivo multiplexed XFI. We envisage that laboratory liquid-jet X-ray technology will significantly contribute to advancing our understanding of biological systems in the field of nanomedical research.

Lipid-based nanoparticles are organic nanostructures constituted of phospholipids and cholesterol, displaying high in vivo biocompatibility. They have been demonstrated as effective nanocarriers for drug delivery and targeting. Mapping liposome distribution is crucial as it enables a precise understanding of delivery kinetics, tissue targeting efficiency, and potential off-target effects. Recently, ruthenium-encapsulated liposomes have shown potential for targeted drug delivery, photodynamic therapy, and optical fluorescence imaging. In the present work, we design Ru(bpy)3-encapsulated liposomes (Ru-Lipo) empowering optical and X-ray fluorescence (XRF) properties for dual mode imaging and demonstrate the passivation role of liposomes over the free Ru(bpy)3 compound. We employ whole-body XRF imaging to map the in vivo biodistribution of Ru-Lipo in mice, enabling tumor detection and longitudinal studies with elemental specificity and resolution down to the sub-millimeter scale. Quantitative XRF computed tomography on extracted organs permits targeting efficiency evaluations. These findings highlight the promising role of XRF imaging in pharmacokinetic studies and theranostic applications for the rapid optimization of drug delivery and assessment of targeting efficiency.

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 enabling different imaging modalities are sought for non-invasive bio-diagnostics. A hybrid material, combining optical and X-ray fluorescence is presented as a bioimaging contrast agent. Core NPs based on metallic rhodium (Rh) have been demonstrated to be potential X-ray Fluorescence Computed Tomography (XFCT) contrast agents. Microwave-assisted hydrothermal method is used for NP synthesis, yielding large-scale NPs within a significantly short reaction time. Rh NP synthesis is performed by using a custom designed sugar ligand (LODAN), constituting a strong reducing agent in aqueous solution, which yields NPs with primary amines as surface functional groups. The amino groups on Rh NPs are used to directly conjugate excitation-independent nitrogen-doped carbon quantum dots (CQDs), which are synthesized through citrate pyrolysis in ammonia solution. CQDs provided the Rh NPs with optical fluorescence properties and improved their biocompatibility, as demonstrated in vitro by Real-Time Cell Analysis (RTCA) on a macrophage cell line (RAW 264.7). The multimodal characteristics of the hybrid NPs are confirmed with confocal microscopy, and X-ray Fluorescence (XRF) phantom experiments.

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.

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.