Nowadays, the interest in the design of particles that combine therapy and diagnosis simultaneously to obtain a theranostic material has increased. One of the most used materials for MRI diagnosis is iron oxide, where clusters of superparamagnetic iron oxide (SPIONs) are noteworthy candidates. These particles are of high interest due to their broad range of applications, such as contrast agents, use in magnetic separation processes, and in hyperthermia therapy, among others. One of the major problems with their use is maintaining superparamagnetism while having the highest magnetization-to-particle ratio. In this work, microwave-assisted synthesis of clusters formed by SPIONs has been investigated. This synthesis strategy allows for significant reduction in the time and energy required to obtain SPION clusters. Also, the magnetization-to-particle ratio has been increased in comparison with single SPIONs. Subsequently, the clusters are coated with amorphous silica using the Stöber method, followed by mesoporous (MS) silica using the atrane method, which offers high and conformal coating homogeneity over the clusters. Surfactant extraction was done using a simple mixture of water, ethanol, and sodium chloride – avoiding the use of other organic solvents. Finally, as a proof of concept, the loading and release of a model molecule were studied to confirm that the SPION-NCs@MS presented in this work have great potential as theranostic agents.

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.

X-ray fluorescence imaging (XFI) is an emerging technique for preclinical studies, characterized by high resolution, specificity, and sensitivity. It relies on nanoparticles (NPs) as contrast agents, which must be constituted of specific elements that match the X-ray source energy for detection. Laboratory liquid metal-jet X-ray sources enable compact in vivo XFI, thereby extending the accessibility of this imaging technique beyond synchrotron facilities.

When designing NPs as contrast agents, biocompatibility is essential for both preclinical and clinical imaging, often requiring a passivating biocompatible coating on the NP surface. The NP cores can provide contrast by their elemental composition, while coating, conjugation, and decoration strategies can add other functionalities and improve biocompatibility.

In this thesis, multifunctional NPs are designed to extend the functionality of XFI contrast agents by incorporating optically fluorescent or magnetically active components: conjugated carbon quantum dots, dye-doped silica shell, and decorated superparamagnetic iron oxide NPs. The designed multifunctional NPs allow correlative and multiscale imaging with complementary techniques such as confocal optical microscopy or magnetic resonance imaging (MRI). Furthermore, these NPs also facilitate more comprehensive studies on NP pharmacokinetics, paving the way for more robust investigations in the field of nanomedicine.

The benefits of multifunctional NPs are demonstrated with two approaches. First, in vivo correlative imaging with MRI and XFI is shown to reduce false positives caused by MRI artifacts in the lungs and abdomen. Second, XFI is employed to enable rapid NP bioengineering, by iteratively improving NP properties and administration strategies for passive tumor targeting. Optical and X-ray fluorescent multifunctional NPs enable the co-localization of NPs at both macroscopic and microscopic levels with XFI and confocal microscopy, correlating NP accumulation in organs with NP-cell interactions. These results highlight the role of XFI in the field of nanomedicine, with potential applications in pharmacokinetics, tumor targeting, treatment monitoring, and the development of medical devices.

Röntgenfluorescensavbildning (RFA) är en växande teknik för prekliniska studier, och karakteriseras av hög upplösning, specificitet och känslighet. RFA använder nanopartiklar (NP:ar) som kontrastmedel, vilket måste innehålla specifika element som matchar röntgenkällans energi. Röntgenkällor med flytande metallstråleteknik möjliggör kompakt in vivo RFA i laboratorier, vilket gör denna avbildningsteknik tillgänglig även utanför synkrotronanläggningar.

Vid utformningen av NP:ar som kontrastmedel är biokompatibilitet avgörande betydelse både för preklinisk och klinisk avbildning, vilket ofta kräver ett passiverande biokompatibelt skikt på NP-ytan. NP-kärnorna kan ge kontrast genom sin grundämnessammansättning, medan beläggnings-, konjugerings- och dekorationsstrategier kan lägga till andra funktionaliteter och förbättra biokompatibiliteten.

I denna avhandling syntetiseras multifunktionella NP:ar för att utöka funktionaliteten hos RFA-kontrastmedel genom att inkorporera optiskt fluorescerande eller magnetiskt aktiva komponenter: konjugerade kolkvantprickar, färgämnesdopat  kiseldioxidskal och dekorerade superparamagnetiska järnoxid NP:ar. De utformade multifunktionella NP:arna möjliggör korrelativ avbildning med kompletterande tekniker som konfokal optisk mikroskopi eller magnetisk resonanstomografi (MR). Dessutom underlättar dessa NP:ar också mer omfattande studier av NP-farmakokinetik, vilket banar väg för bättre underbyggda undersökningar inom nanomedicin.

Fördelarna med multifunktionella NP:ar demonstreras med två tillvägagångssätt. För det första har in vivo korrelativ avbildning med MR och RFA visat sig minska antalet falska positiva resultat orsakade av MR-artefakter i lungorna och buken. För det andra används RFA för att möjliggöra snabb utveckling och design av NP:ar, genom att iterativt förbättra NP-egenskaper och administreringsstrategier för passiv ansamling i tumörer. Optiska och röntgenfluorescerande multifunktionella NP:ar möjliggör samlokalisering av NP:ar på både makroskopisk och mikroskopisk nivå med RFA och konfokal mikroskopi, vilket korrelerar NP-ackumuleringar i organ med NP-cellinteraktioner. Dessa resultat belyser RFA:s roll inom nanomedicinfältet, med dess potentiella tillämpningar inom farmakokinetik, tumörmålsökning, behandlingsövervakning och utveckling av medicinska instrument.

Nowadays, superparamagnetic iron oxide nanoparticles (SPIONs) have a dominant role in many subfields of biomedicine. Owing to their peculiar properties, they can be employed for magnetic separation, drug delivery, diagnostics, and hyperthermia treatments. However, these magnetic nanoparticles (NPs) suffer from low unit magnetization due to size constraints (up to 20-30 nm) to exhibit superparamagnetic character. In this work, we have designed and synthesized superparamagnetic nanoclusters (SP-NCs) with diameters of up to 400 nm with high unit magnetization for enhanced loading capacity. These were synthesized with conventional or microwave-assisted solvothermal methods, in the presence of either of the two biomolecules (citrate or l-lysine) as the capping agent. Primary particle size, SP-NC size, surface chemistry, and the resultant magnetic properties were observed to be significantly influenced by the choice of synthesis route and capping agent. Selected SP-NCs were then coated with a fluorophore-doped silica shell to provide fluorescence properties, in the near-infrared spectrum region, while silica provided high chemical and colloidal stability. Heating efficiency studies were performed under alternating magnetic field on the synthesized SP-NCs, highlighting their potential in hyperthermia treatment. We envision that their enhanced magnetically-active content, fluorescence, magnetic property, and heating efficiency will pave the way to more effective uses in biomedical applications.

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.

Two-photon polymerization (2PP) is an efficient technique to achieve high-resolution, three-dimensional (3D)-printed complex structures. However, it is restricted to photocurable monomer combinations, thus presenting constraints when aiming at attaining functionally active resist formulations and structures. In this context, metal nanoparticle (NP) integration as an additive can enable functionality and pave the way to more dedicated applications. Challenges lay on the maximum NP concentrations that can be incorporated into photocurable resist formulations due to the laser-triggered interactions, which primarily originate from laser scattering and absorption, as well as the limited dispersibility threshold. In this study, we propose an approach to address these two constraints by integrating metallic Rh NPs formed ex situ, purposely designed for this scope. The absence of surface plasmon resonance (SPR) within the visible and near-infrared spectra, coupled with the limited absorption value measured at the laser operating wavelength (780 nm), significantly limits the laser-induced interactions. Moreover, the dispersibility threshold is increased by engineering the NP surface to be compatible with the photocurable resin, permitting us to achieve concentrations of up to 2 wt %, which, to our knowledge, is significantly higher than the previously reported limit (or threshold) for embedded metal NPs. Another distinctive advantage of employing Rh NPs is their role as promising contrast agents for X-ray fluorescence (XRF) bioimaging. We demonstrated the presence of Rh NPs within the whole 2PP-printed structure and emphasized the potential use of NP-loaded 3D-printed nanostructures for medical devices.

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.

For the time being, a great attention has been given to the search of green and reusable materials with antibacterial properties. The present research focused on the design and synthesis of hybrid structures constituting superparamagnetic iron oxide nanoparticles (SPIONs) coated with sugar ligands (SL), synthesized using a green and efficient microwave (MW)-assisted hydrothermal synthesis. The sugar ligands were selectively engineered to obtain antibacterial characteristics towards multi-drug resistant bacterial strains, which are among the most problematic bacterial species in antibiotic development efforts. The superparamagnetic behavior was obtained by synthesizing core iron oxide nanoparticles with a diameter below twenty nm. The MW-assisted hydrothermal method yielded a uniform coating of SPIONs with several sugar ligands, granting strongly negative-charged surfaces, which have eventually contributed to their bactericidal activity. The research work allowed to get insights into the magnetic properties of the sugar ligand coated SPIONs, as well as on morphological and functional characteristics of the hybrid nanoparticles, by employing both spectroscopy and imaging techniques, such as FT-IR, Scanning/Transmission Electron Microscopy (S/TEM). Detailed characterizations of the nanoparticles' charge, using zeta potential analysis helped to identify the highly charged hybrids for antibacterial applications. Furthermore, studies on the bactericidal properties of selected SL-SPION hybrids highlighted a high selectivity towards both gram-negative and gram-positive bacteria along with improving bactericidal activity of streptomycin/penicillin mixture. Detailed studies done on Pseudomonas aeruginosa revealed that the SPIONs selectively downregulated the virulence factor pyoverdine and altered bacterial morphology depending on the SL chemistry. The synthesized materials with antibacterial activity pave the way for an effective path towards the design and development of nanostructures and coatings against antibiotic-resistant bacterial species.