Functionalized Lipopeptide Micelles as Highly Efficient NMR Depolarization Seed Points for Targeted Cell Labelling in Xenon MRI.

Improving diagnostic imaging and therapy by targeted compound delivery to pathological areas and across biological barriers is of urgent need. A lipopeptide, P-CrA-A2, composed of a highly cationic peptide sequence (A2), an N-terminally attached palmitoyl chain (P) and cryptophane molecule (CrA) for preferred uptake into blood-brain barrier (BBB) capillary endothelial cells, was generated. CrA allows reversible binding of Xe for NMR detection with hyperpolarized nuclei. The lipopeptide forms size-optimized micelles with a diameter of about 11 nm at low micromolar concentration. Their high local CrA payload has a strong and switchable impact on the bulk magnetization through Hyper-CEST detection. Covalent fixation of CrA does not impede micelle formation and does not hamper its host functionality but simplifies Xe access to hosts for inducing saturation transfer. Xe Hyper-CEST magnetic resonance imaging (MRI) allows for distinguishing BBB endothelial cells from control aortic endothelial cells, and the small micelle volume with a sevenfold improved CrA-loading density compared to liposomal carriers allows preferred cell labelling with a minimally invasive volume (≈16 000-fold more efficient than 19 F cell labelling). Thus, these nanoscopic particles combine selectivity for human brain capillary endothelial cells with great sensitivity of Xe Hyper-CEST MRI and might be a potential MRI tool in brain diagnostics.

High Exchange Rate Complexes of 129 Xe with Water-Soluble Pillar[5]arenes for Adjustable Magnetization Transfer MRI.

Macrocyclic host structures for generating transiently bound 129 Xe have been used in various ultra-sensitive NMR and MRI applications for molecular sensing of biochemical analytes. They are based on hyperpolarized nuclei chemical exchange saturation transfer (Hyper-CEST). Here, we tested a set of water-soluble pillar[5]arenes with different counterions in order to compare their potential contrast agent abilities with that of cryptophane-A (CrA), the most widely used host for such purposes. The exchange of Xe with such compounds was found to be sensitive to the type of ions present in solution and can be used for switchable magnetization transfer (MT) contrast that arises from off-resonant pre-saturation. We demonstrate that the adjustable MT magnitude depends on the interplay of saturation parameters and found that the optimum MT contrast surpasses the CrA CEST performance at moderate saturation power. Since modification of such water-soluble pillar[5]arenes is straightforward, these compounds can be considered a promising platform for designing various sensors that may complement the field of Xe HyperCEST-based biosensing MRI.

Time-resolved monitoring of enzyme activity with ultrafast Hyper-CEST spectroscopy.

We propose a method to dynamically monitor the progress of an enzymatic reaction using NMR of hyperpolarized 129 Xe in a host-guest system. It is based on a displacement assay originally designed for fluorescence experiments that exploits the competitive binding of the enzymatic product on the one hand and a reporter dye on the other hand to a supramolecular host. Recently, this assay has been successfully transferred to NMR, using xenon as a reporter, cucurbit[6]uril as supramolecular host, and chemical exchange saturation transfer with hyperpolarized Xe (Hyper-CEST) as detection technique. Its advantage is that the enzyme acts on the unmodified substrate and that only the product is detected through immediate inclusion into the host. We here apply a method that drastically accelerates the acquisition of Hyper-CEST spectra in vitro using magnetic field gradients. This allows monitoring the dynamic progress of the conversion of lysine to cadaverine with a temporal resolution of ~30 s. Moreover, the method only requires to sample the very early onset of the reaction (<0.5% of substrate conversion where the host itself is required only at µM concentrations) at comparatively low reaction rates, thus saving enzyme material and reducing NMR acquisition time. The obtained value for the specific activity agrees well with previously published results from fluorescence assays. We furthermore outline how the Hyper-CEST results correlate with xenon T2 measurements performed during the enzymatic reaction. This suggests that ultrafast Hyper-CEST spectroscopy can be used for dynamically monitoring enzymatic activity with NMR.

Supramolecular Assays for Mapping Enzyme Activity by Displacement-Triggered Change in Hyperpolarized (129)Xe Magnetization Transfer NMR Spectroscopy.

Reversibly bound Xe is a sensitive NMR and MRI reporter with its resonance frequency being influenced by the chemical environment of the host. Molecular imaging of enzyme activity presents a promising approach for disease identification, but current Xe biosensing concepts are limited since substrate conversion typically has little impact on the chemical shift of Xe inside tailored cavities. Herein, we exploit the ability of the product of the enzymatic reaction to bind itself to the macrocyclic hosts CB6 and CB7 and thereby displace Xe. We demonstrate the suitability of this method to map areas of enzyme activity through changes in magnetization transfer with hyperpolarized Xe under different saturation scenarios.

Brain endothelial cell targeting via a peptide-functionalized liposomal carrier for xenon hyper-CEST MRI.

A nanoparticulate carrier system is used to efficiently deliver a contrast agent for highly sensitive xenon Hyper-CEST MRI. The carrier system not only improves the biocompatibility and solubility of the contrast agent, it also allows selective cell targeting as demonstrated by the discrimination of human brain capillary and aortic endothelial cells.

Depolarization Laplace transform analysis of exchangeable hyperpolarized ¹²9Xe for detecting ordering phases and cholesterol content of biomembrane models.

We present a highly sensitive nuclear-magnetic resonance technique to study membrane dynamics that combines the temporary encapsulation of spin-hyperpolarized xenon ((129)Xe) atoms in cryptophane-A-monoacid (CrAma) and their indirect detection through chemical exchange saturation transfer. Radiofrequency-labeled Xe@CrAma complexes exhibit characteristic differences in chemical exchange saturation transfer-driven depolarization when interacting with binary membrane models composed of different molecular ratios of DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine). The method is also applied to mixtures of cholesterol and POPC. The existence of domains that fluctuate in cluster size in DPPC/POPC models at a high (75-98%) DPPC content induces up to a fivefold increase in spin depolarization time τ at 297 K. In POPC/cholesterol model membranes, the parameter τ depends linearly on the cholesterol content at 310 K and allows us to determine the cholesterol content with an accuracy of at least 5%.

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.

Functionalized 129Xe as a potential biosensor for membrane fluidity.

Using spin hyperpolarized xenon ((129)Xe) we investigate the impact of the local molecular environment on reversible host-guest interactions. We label Xe guest atoms that are temporarily bound to cryptophane-A hosts using the Hyper-CEST technique. By varying the length of the saturation pulse and utilizing an inverse Laplace transform we can determine depolarization times for the noble gas in different local environments, in this case biomembranes possessing different fluidity. We extend this technique to magnetic resonance imaging, mapping the spatial distribution of the different biomembranes. Such decays measured in biomembranes of 200 µM 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were characterized by mono-exponential decays with time constants of τPOPC = 3.00(-0.61)(+0.77) s and τDPPC(-4.16)(+5.19) = 22.15 s. Analyzing both environments simultaneously yielded a bi-exponential decay. This approach may give further insights into saturation transfer dynamics of reversibly bound Xe with applications extending into biomedical diagnostics.

Biomembrane interactions of functionalized cryptophane-A: combined fluorescence and 129Xe NMR studies of a bimodal contrast agent.

Fluorescent derivatives of the (129)Xe NMR contrast agent cryptophane-A were obtained by functionalization with near infrared fluorescent dyes DY680 and DY682. The resulting conjugates were spectrally characterized, and their interaction with giant and large unilamellar vesicles of varying phospholipid composition was analyzed by fluorescence and NMR spectroscopy. In the latter, a chemical exchange saturation transfer with hyperpolarized (129)Xe (Hyper-CEST) was used to obtain sufficient sensitivity. To determine the partitioning coefficients, we developed a method based on fluorescence resonance energy transfer from Nile Red to the membrane-bound conjugates. This indicated that not only the hydrophobicity of the conjugates, but also the phospholipid composition, largely determines the membrane incorporation. Thereby, partitioning into the liquid-crystalline phase of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine was most efficient. Fluorescence depth quenching and flip-flop assays suggest a perpendicular orientation of the conjugates to the membrane surface with negligible transversal diffusion, and that the fluorescent dyes reside in the interfacial area. The results serve as a basis to differentiate biomembranes by analyzing the Hyper-CEST signatures that are related to membrane fluidity, and pave the way for dissecting different contributions to the Hyper-CEST signal.

Analytical solution for the depolarization of hyperpolarized nuclei by chemical exchange saturation transfer between free and encapsulated xenon (HyperCEST).

We present an analytical solution of the Bloch-McConnell equations for the case of chemical exchange saturation transfer between hyperpolarized nuclei in cavities and in solvent (HyperCEST experiment). This allows quantitative investigation of host-guest interactions by means of nuclear magnetic resonance spectroscopy and, due to the strong HyperCEST signal enhancement, even NMR imaging. Hosts of interest can be hydrophobic cavities in macromolecules or artificial cages like cryptophane-A which was proposed as a targeted biosensor. Relevant system parameters as exchange rate and host concentration can be obtained from the monoexponential depolarization process which is shown to be governed by the smallest eigenvalue in modulus. For this dominant eigenvalue we present a useful approximation leading to the depolarization rate for the case of on- and off-resonant irradiation. It is shown that this rate is a generalization of the longitudinal relaxation rate in the rotating frame. We demonstrate for the free and cryptophane-A-encapsulated xenon system, by comparison with numerical simulations, that HyperCEST experiments are precisely described in the valid range of this widely applicable analytical approximation. Altogether, the proposed analytical solution allows optimization and quantitative analysis of HyperCEST experiments but also characterization and optimal design of possible biosensors.