Probing Reversible Guest Binding with Hyperpolarized 129Xe-NMR: Characteristics and Applications for Cucurbit[n]urils.

Cucurbit[n]urils (CB[n]s) are a family of macrocyclic host molecules that find various applications in drug delivery, molecular switching, and dye displacement assays. The CB[n]s with n = 5-7 have also been studied with 129Xe-NMR. They bind the noble gas with a large range of exchange rates. Starting with insights from conventional direct detection of bound Xe, this review summarizes recent achievements with chemical exchange saturation transfer (CEST) detection of efficiently exchanging Xe in various CB[n]-based supramolecular systems. Unprecedented sensitivity has been reached by combining the CEST method with hyperpolarized Xe, the production of which is also briefly described. Applications such as displacement assays for enzyme activity detection and rotaxanes as emerging types of Xe biosensors are likewise discussed in the context of biomedical applications and pinpoint future directions for translating this field to preclinical studies.

Molecular Sensing with Host Systems for Hyperpolarized 129Xe.

Hyperpolarized noble gases have been used early on in applications for sensitivity enhanced NMR. 129Xe has been explored for various applications because it can be used beyond the gas-driven examination of void spaces. Its solubility in aqueous solutions and its affinity for hydrophobic binding pockets allows "functionalization" through combination with host structures that bind one or multiple gas atoms. Moreover, the transient nature of gas binding in such hosts allows the combination with another signal enhancement technique, namely chemical exchange saturation transfer (CEST). Different systems have been investigated for implementing various types of so-called Xe biosensors where the gas binds to a targeted host to address molecular markers or to sense biophysical parameters. This review summarizes developments in biosensor design and synthesis for achieving molecular sensing with NMR at unprecedented sensitivity. Aspects regarding Xe exchange kinetics and chemical engineering of various classes of hosts for an efficient build-up of the CEST effect will also be discussed as well as the cavity design of host molecules to identify a pool of bound Xe. The concept is presented in the broader context of reporter design with insights from other modalities that are helpful for advancing the field of Xe biosensors.

Complete Generation of a 129Xe Biosensor on the Solid Support by Systematic Backbone Assembly.

Xenon biosensors are an emerging tool for different molecular imaging approaches. For many applications, their development requires peptide synthesis steps, followed by the selective installation of a xenon host onto the peptide backbone in solution. In this study, three different strategies were attempted for generating entire Xe biosensors on the solid support. Notably, one strategy involving CryA-da was beneficial by directly integrating this host into the growing construct on a low loaded resin via modification of the administered subcomponent equivalents and by prolonging the coupling procedure. Subsequently, installation of additional amino acids or of additional labels onto the growing construct was achieved by a procedure in which an excess amine was administered to the activated CryA-da (acid) anchored onto the resin. Further, the as-generated Xe biosensor was tested for its NMR and MRI capabilities in H2O and compared to the performance of CryA-ma. Xe NMR of the biosensor indicated a clear CEST response and the Xe MR images revealed similar contrast compared to the reference host. These observations suggest that functionalizing CryA-da on both sides with multiple labels did not alter significantly its NMR capabilities. Hereby, we could show the successful and complete synthesis of a CryA-da-based xenon biosensor on the solid support without any notable side reactions and without the necessity of multiple purification steps.

Cell tracking with caged xenon: using cryptophanes as MRI reporters upon cellular internalization.

Caged xenon has great potential in overcoming sensitivity limitations for solution-state NMR detection of dilute molecules. However, no application of such a system as a magnetic resonance imaging (MRI) contrast agent has yet been performed with live cells. We demonstrate MRI localization of cells labeled with caged xenon in a packed-bed bioreactor working under perfusion with hyperpolarized-xenon-saturated medium. Xenon hosts enable NMR/MRI experiments with switchable contrast and selectivity for cell-associated versus unbound cages. We present MR images with 10(3) -fold sensitivity enhancement for cell-internalized, dual-mode (fluorescence/MRI) xenon hosts at low micromolar concentrations. Our results illustrate the capability of functionalized xenon to act as a highly sensitive cell tracer for MRI detection even without signal averaging. The method will bridge the challenging gap for translation to in vivo studies for the optimization of targeted biosensors and their multiplexing applications.

Hyper-CEST NMR of metal organic polyhedral cages reveals hidden diastereomers with diverse guest exchange kinetics.

Guest capture and release are important properties of self-assembling nanostructures. Over time, a significant fraction of guests might engage in short-lived states with different symmetry and stereoselectivity and transit frequently between multiple environments, thereby escaping common spectroscopy techniques. Here, we investigate the cavity of an iron-based metal organic polyhedron (Fe-MOP) using spin-hyperpolarized 129Xe Chemical Exchange Saturation Transfer (hyper-CEST) NMR. We report strong signals unknown from previous studies that persist under different perturbations. On-the-fly delivery of hyperpolarized gas yields CEST signatures that reflect different Xe exchange kinetics from multiple environments. Dilute pools with ~ 104-fold lower spin numbers than reported for directly detected hyperpolarized nuclei are readily detected due to efficient guest turnover. The system is further probed by instantaneous and medium timescale perturbations. Computational modeling indicates that these signals originate likely from Xe bound to three Fe-MOP diastereomers (T, C3, S4). The symmetry thus induces steric effects with aperture size changes that tunes selective spin manipulation as it is employed in CEST MRI agents and, potentially, impacts other processes occurring on the millisecond time scale.

RGD-labeled USPIO inhibits adhesion and endocytotic activity of alpha v beta3-integrin-expressing glioma cells and only accumulates in the vascular tumor compartment.

PURPOSE: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Nanoparticle-Based Contrast Agents for 129Xe HyperCEST NMR and MRI Applications.

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 with 129Xe 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.

In vivo evaluation of riboflavin receptor targeted fluorescent USPIO in mice with prostate cancer xenografts.

Riboflavin (Rf) receptors bind and translocate Rf and its phosphorylated forms (e.g. flavin mononucleotide, FMN) into cells where they mediate various cellular metabolic pathways. Previously, we showed that FMN-coated ultrasmall superparamagnetic iron oxide (FLUSPIO) nanoparticles are suitable for labeling metabolically active cancer and endothelial cells in vitro. In this study, we focused on the in vivo application of FLUSPIO using prostate cancer xenografts. Size, charge, and chemical composition of FLUSPIO were evaluated. We explored the in vitro specificity of FLUSPIO for its cellular receptors using magnetic resonance imaging (MRI) and Prussian blue staining. Competitive binding experiments were performed in vivo by injecting free FMN in excess. Bio-distribution of FLUSPIO was determined by estimating iron content in organs and tumors using a colorimetric assay. AFM analysis and zeta potential measurements revealed a particulate morphology approximately 20-40 nm in size and a negative zeta potential (-24.23 ± 0.15 mV) in water. X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry data confirmed FMN present on the USPIO nanoparticle surface. FLUSPIO uptake in prostate cancer cells and human umbilical vein endothelial cells was significantly higher than that of control USPIO, while addition of excess of free FMN reduced accumulation. Similarly, in vivo MRI and histology showed specific FLUSPIO uptake by prostate cancer cells, tumor endothelial cells, and tumor-associated macrophages. Besides prominent tumor accumulation, FLUSPIO accumulated in the liver, spleen, lung, and skin. Hence, our data strengthen our hypothesis that targeting riboflavin receptors is an efficient approach to accumulate nanomedicines in tumors opening perspectives for the development of diagnostic and therapeutic systems. ELECTRONIC SUPPLEMENTARY MATERIAL: Supplementary material is available for this article at 10.1007/s12274-016-1028-7 and is accessible for authorized users.

Refinement of adsorptive coatings for fluorescent riboflavin-receptor-targeted iron oxide nanoparticles.

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.

In Vivo Nanotoxicity Testing using the Zebrafish Embryo Assay.

Nanoparticles are increasingly used for biomedical purposes. Many different diagnostic and therapeutic applications are envisioned for nanoparticles, but there are often also serious concerns regarding their safety. Given the fact that numerous new nanomaterials are being developed every day, and that not much is known about the long-term toxicological impact of exposure to nanoparticles, there is an urgent need to establish efficient methods for nanotoxicity testing. The zebrafish (Danio rerio) embryo assay has recently emerged as an interesting 'intermediate' method for in vivo nanotoxicity screening, enabling (semi-) high-throughput analyses in a system significantly more complex than cultured cells, but at the same time also less 'invasive' and less expensive than large-scale biocompatibility studies in mice or rats. The zebrafish embryo assay is relatively well-established in the environmental sciences, but it has not yet gained wide notice in the nanomedicine field. Using prototypic polymeric drug carriers, gold-based nanodiagnostics and nanotherapeutics, and iron oxide-based nanodiagnostics, we here show that toxicity testing using zebrafish embryos is easy, efficient and informative, and faithfully reflects, yet significantly extends, cell-based toxicity testing. We therefore expect that the zebrafish embryo assay will become a popular future tool for in vivo nanotoxicity screening.

Riboflavin carrier protein-targeted fluorescent USPIO for the assessment of vascular metabolism in tumors.

Riboflavin (Rf) and its metabolic analogs flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are essential for normal cellular growth and function. Their intracellular transport is regulated by the riboflavin carrier protein (RCP), which has been shown to be over-expressed by metabolically active cancer cells. Therefore, FAD-decorated ultrasmall superparamagnetic iron oxide nanoparticles (FAD USPIO) were developed as the first carrier-protein-targeted molecular MR agents for visualizing tumor metabolism. FAD USPIO were synthesized using an adsorptive, fluorescent and non-polymeric coating method, and their physicochemical properties were characterized using TEM, SEM, FTIR, MRI and fluorescence spectroscopy. In vitro analyses showed the biocompatibility of FAD USPIO, and confirmed that they were strongly and specifically taken up by cancer (LnCap) and endothelial (HUVEC) cells. In vivo molecular MRI together with subsequent histological validation finally demonstrated that FAD USPIO efficiently accumulate in tumors and tumor blood vessels, indicating that RCP-targeted diagnostic nanoparticles are interesting new materials for the assessment of vascular metabolism in tumors.

Theranostic systems and strategies for monitoring nanomedicine-mediated drug targeting.

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.

Fluorescent magnetoliposomes as a platform technology for functional and molecular MR and optical imaging.

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.

FMN-coated fluorescent iron oxide nanoparticles for RCP-mediated targeting and labeling of metabolically active cancer and endothelial cells.

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 T(2)-relaxivity, are favorably suited for MR cell tracking experiments and cancer detection in vivo.