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Spin hyperpolarization techniques have enabled important advancements in preclinical and clinical MRI applications to overcome the intrinsic low sensitivity of nuclear magnetic resonance. Functionalized xenon biosensors represent one of these approaches. They combine two amplification strategies, namely, spin exchange optical pumping (SEOP) and chemical exchange saturation transfer (CEST). The latter one requires host structures that reversibly bind the hyperpolarized noble gas. Different nanoparticle approaches have been implemented and have enabled molecular MRI with129Xe at unprecedented sensitivity. This review gives an overview of the Xe biosensor concept, particularly how different nanoparticles address various critical aspects of gas binding and exchange, spectral dispersion for multiplexing, and targeted reporter delivery. As this concept is emerging into preclinical applications, comprehensive sensor design will be indispensable in translating the outstanding sensitivity potential into biomedical molecular imaging applications.

Riboflavin is an essential vitamin for cellular metabolism and is highly upregulated in metabolically active cells. Consequently, targeting the riboflavin carrier protein (RCP) may be a promising strategy for labeling cancer and activated endothelial cells. Therefore, Ultrasmall SuperParamagnetic Iron Oxide nanoparticles (USPIO) were adsorptively coated with the endogenous RCP ligand flavin mononucleotide (FMN), which renders them target-specific and fluorescent. The core diameter, surface morphology and surface coverage of the resulting FMN-coated USPIO (FLUSPIO) were evaluated using a variety of physico-chemical characterization techniques (TEM, DLS, MRI and fluorescence spectroscopy). The biocompatibility of FLUSPIO was confirmed using three different cell viability assays (Trypan blue staining, 7-AAD staining and TUNEL). In vitro evaluation of FLUSPIO using MRI and fluorescence microscopy demonstrated high labeling efficiency of cancer cells (PC-3, DU-145, LnCap) and activated endothelial cells (HUVEC). Competition experiments (using MRI and ICP-MS) with a 10- and 100-fold excess of free FMN confirmed RCP-specific uptake of the FLUSPIO by PC-3 cells and HUVEC. Hence, RCP-targeting via FMN may be an elegant way to render nanoparticles fluorescent and to increase the labeling efficacy of cancer and activated endothelial cells. This was shown for FLUSPIO, which due to their high T2-relaxivity, are favorably suited for MR cell tracking experiments and cancer detection in vivo.

To investigate the biologic effect of arginine-glycine-aspartic acid (RGD)-labeled ultrasmall superparamagnetic iron oxide (USPIO) (referred to as RGD-USPIO) on human umbilical vein endothelial cells (HUVECs), ovarian carcinoma (MLS) cells, and glioblastoma (U87MG) cells and on U87MG xenografts in vivo.All experiments were approved by the governmental review committee on animal care.USPIOs were coated with integrin-specific (RGD) or unspecific (arginine-alanine-aspartic acid [RAD]) peptides. USPIO uptake in HUVECs, MLS cells, and U87MG cells and in U87MG tumor xenografts was determined with T2 magnetic resonance (MR) relaxometry in 16 nude mice. Cells and tumors were characterized by using immunofluorescence microscopy. Trypan blue staining and lactate dehydrogenase assay were used to assess cytotoxicity. Statistical evaluation was performed by using a Mann-Whitney test or a linear mixed model with random intercept for the comparison of data from different experiments. Post hoc pairwise comparisons were adjusted according to a Tukey test.HUVECs and MLS cells internalized RGD-USPIOs significantly more than unspecific probes. Controversially, U87MG cells accumulated RGD-USPIOs to a lesser extent than USPIO. Furthermore, only in U87MG cells, free RGD and alpha(v)beta(3) integrin-blocking antibodies strongly reduced endocytosis of nonspecific USPIOs. This was accompanied by a loss of cadherin-dependent intercellular contacts, which could not be attributed to cell damage. In U87MG tumors, RGD-USPIO accumulated exclusively at the neovasculature but not within tumor cells. The vascular accumulation of RGD-USPIO caused significantly higher changes of the R2 relaxation rate of tumors than observed for USPIO.In glioma cells with unstable intercellular contacts, inhibition of alpha(v)beta(3) integrins by antibodies and RGD and RGD-USPIO disintegrated intercellular contacts and reduced endocytotic activity, illustrating the risk of inducing biologic effects by using molecular MR probes.

Flavin mononucleotide (FMN) is a riboflavin derivative that can be exploited to target the riboflavin transporters (RFTs) and the riboflavin carrier protein (RCP) in cells with high metabolic activity. In this study we present the synthesis of different FMN-coated ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs) and their efficiency as targeting contrast agents. Since FMN alone cannot stabilize the nanoparticles, we used adenosine phosphates--AMP, ADP and ATP--as spacers to obtain colloidally stable nanoparticles. Nucleotides with di- and triphosphate groups were intended to increase the USPIO charge and thus improve zeta potential and stability. However, all nanoparticles formed negatively charged clusters with similar properties in terms of zeta potential (-28 ± 2 mV), relaxivity (228-259 mM(-1) s(-1) at 3 T) and hydrodynamic radius (53-85 nm). Molecules with a higher number of phosphate groups, such as ADP and ATP, have a higher adsorption affinity towards iron oxide, which, instead of providing more charge, led to partial desorption and replacement of FMN. Hence, we obtained USPIOs carrying different amounts of targeting agent, which significantly influenced the nanoparticles' uptake. The nanoparticles' uptake by different cancer cells and HUVECs was evaluated photometrically and with MR relaxometry, showing that the cellular uptake of the USPIOs increases with the FMN amount on their surface. Thus, for USPIOs targeted with riboflavin derivatives the use of spacers with increasing numbers of phosphate groups does not improve either zeta potential or the particles' stability, but rather detaches the targeting moieties from their surface, leading to lower cellular uptake.

Here, we present a detailed characterisation of rhodamine B-containing magnetoliposomes (FLU-ML), emphasising the dependence of their fluorescence properties on the presence of iron oxide cores, and the molar fraction of the fluorophore. The magnetoliposome types used exist as colloidally stable, negatively charged clusters with an average hydrodynamic diameter of 95 nm. The molar rhodamine B fractions were 0.67 % and 1.97 %. Rhodamine B normalised fluorescence, quantum yields and fluorescence lifetimes were substantially reduced by inner filter effects as the magnetoliposome concentration is increased, by increasing molar rhodamine B fraction, and by quenching originating from the iron oxide cores. MR relaxometry at 3 T revealed extremely high r2 relaxivities (440 to 554 s-1mM-1) and moderately high r1 values (2.06 to 3.59 s-1mM-1). Upon incubating human prostate carcinoma (PC-3) cells with FLU-ML, a dose-dependent particle internalisation was found by MR relaxometry. In addition, the internalised FLU-ML were clearly visible by fluorescence microscopy. At the FLU-ML concentrations used (up to 3 × 10³ M Fe) cell viability was not substantially impaired. These results provide valuable insights on the fluorescence properties of bimodal magnetoliposomes and open promising perspectives for the use of these materials as a platform technology for advanced functional and molecular MR and optical imaging applications.

Nanomedicine formulations are considered to be superior to standard low-molecular-weight drugs because of an increased drug accumulation at the pathological site and a decreased localization to healthy non-target tissues, together leading to an improved balance between the efficacy and the toxicity of (chemo-) therapeutic interventions. To better understand and further improve nanomedicine-mediated drug targeting, it is important to design systems and strategies which are able to provide real-time feedback on the localization, the release and the therapeutic efficacy of these formulations. The advances made over the past few years with regard to the development of novel imaging agents and techniques have provided a broad basis for the design of theranostic nanomedicine materials, i.e. multicomponent carrier constructs in which drugs and imaging agents are combined, and which can be used to address issues related to drug localization, drug release and drug efficacy. Here, we summarize several recent efforts in this regard, and we show that theranostic systems and strategies hold significant potential for monitoring and improving nanomedicine-mediated drug targeting.