Paramagnetic Agent Research Articles

Using frequency‐labeled exchange transfer to separate out conventional magnetization transfer effects from exchange transfer effects when detecting ParaCEST agents

Paramagnetic chemical exchange saturation transfer agents combine the benefits of a large chemical shift difference and a fast exchange rate for sensitive MRI detection. However, the in vivo detection of these agents is hampered by the need for high B(1) fields to allow sufficiently fast saturation before exchange occurs, thus causing interference of large magnetization transfer effects from semisolid macromolecules. A recently developed approach named frequency-labeled exchange transfer utilizes excitation pulses instead of saturation pulses for detecting the exchanging protons. Using solutions and gel phantoms containing the europium (III) complex of DOTA tetraglycinate (EuDOTA-(gly)(-) (4) ), it is shown that frequency-labeled exchange transfer allows the separation of chemical exchange effects and magnetization transfer (MT) effects in the time domain, therefore allowing the study of the individual resonance of rapidly exchanging water molecules (k(ex) >10(4) s(-1) ) without interference from conventional broad-band MT.

3D‐liver perfusion MRI with the MS‐325 blood pool agent: A noninvasive protocol to asses liver fibrosis

To evaluate a 1.5T magnetic resonance imaging (MRI) protocol, including a dedicated acquisition sequence and a postprocessing tool for the quantitative analysis of hepatic tissue perfusion. Estimated perfusion parameters and histological results based on the METAVIR classification were prospectively compared for hepatic fibrosis assessment. The study protocol was approved by the experimentation Ethics Committee and informed consent was obtained. Sixteen patients (6 women, 10 men; average age, 52.4 ± 14.8 years) with chronic liver diseases were prospectively enrolled after a liver biopsy. MS-325 (paramagnetic blood pool agent)-enhanced MRI was performed using a free-breath 3D-VIBE T(1w) sequence. Image volumes were registered by an automatic rigid method. Liver perfusion was modeled by a dual-input-one-compartment model and quantitative perfusion parameters such as arterial, portal, and total perfusion mean transit time (MTT) and hepatic perfusion index (HPI) were obtained using in-house developed software. Arterial perfusion increased with METAVIR stage, whereas portal perfusion decreased leading to an HPI increase with fibrosis stage. MTT increased with F3, F4. A nonparametric Mann-Whitney test demonstrated that HPI and portal perfusion were relevant in discriminating between advanced and nonadvanced fibrosis, between fibrosis and cirrhosis, then between nonfibrosis and fibrosis (P < 0.01). A strong correlation was found between portal perfusion fall-off and HPI increase (r = -0.97; P < 0.001). HPI and portal perfusion were strongly correlated with fibrosis stage (r = 0.83 and -0.88; P < 0.001, respectively). HPI and portal perfusion could be relevant indicators for the clinical follow-up in patients with chronic liver diseases.

Structural Characterization of Hsp12, the Heat Shock Protein from Saccharomyces cerevisiae, in Aqueous Solution Where It Is Intrinsically Disordered and in Detergent Micelles Where It Is Locally α-Helical

Hsp12 (heat shock protein 12) belongs to the small heat shock protein family, partially characterized as a stress response, stationary phase entry, late embryonic abundant-like protein located at the plasma membrane to protect membrane from desiccation. Here, we report the structural characterization of Hsp12 by NMR and biophysical techniques. The protein was labeled uniformly with nitrogen-15 and carbon-13 so that its conformation could be determined in detail both in aqueous solution and in two membrane-mimetic environments, SDS and dodecylphosphocholine (DPC) micelles. Secondary structural elements determined from assigned chemical shifts indicated that Hsp12 is dynamically disordered in aqueous solution, whereas it gains four helical stretches in the presence of SDS micelles and a single helix in presence of DPC. These conclusions were reinforced by circular dichroism spectra of the protein in all three environments. The lack of long range interactions in NOESY spectra indicated that the helices present in SDS micelles do not pack together. R(1) and R(2), relaxation and heteronuclear NOE measurements showed that the protein is disordered in aqueous solution but becomes more ordered in presence of detergent micelles. NMR spectra collected in presence of paramagnetic spin relaxation agents (5DSA, 16DSA, and Gd(DTPA-BMA)) indicated that the amphipathic α-helices of Hsp12 in SDS micelles lie on the membrane surface. These observations are in agreement with studies suggesting that Hsp12 functions to protect the membrane from desiccation.

Sensitizing solid state nuclear magnetic resonance of dilute nuclei by spin-diffusion assisted polarization transfers

Recent years have witnessed efforts geared at increasing the sensitivity of NMR experiments, by relying on the suitable tailoring and exploitation of relaxation phenomena. These efforts have included the use of paramagnetic agents, enhanced (1)H-(1)H incoherent and coherent transfers processes in 2D liquid state spectroscopy, and homonuclear (13)C-(13)C spin diffusion effects in labeled solids. The present study examines some of the opportunities that could open when exploiting spontaneous (1)H-(1)H spin-diffusion processes, to enhance relaxation and to improve the sensitivity of dilute nuclei in solid state NMR measurements. It is shown that polarization transfer experiments executed under sufficiently fast magic-angle-spinning conditions, enable a selective polarization of the dilute low-γ spins by their immediate neighboring protons. Repolarization of the latter can then occur during the time involved in monitoring the signal emitted by the low-γ nuclei. The basic features involved in the resulting approach, and its potential to improve the effective sensitivity of solid state NMR measurements on dilute nuclei, are analyzed. Experimental tests witness the advantages that could reside from utilizing this kind of approach over conventional cross-polarization processes. These measurements also highlight a number of limitations that will have to be overcome for transforming selective polarization transfers of this kind into analytical methods of choice.

Fluorescent and Paramagnetic Chitosan Nanoparticles that Exhibit High Magnetic Resonance Relaxivity: Synthesis, Characterization and &lt;I&gt;In Vitro&lt;/I&gt; Studies

We report water-in-oil (W/O) microemulsion synthesis of fluorescently bright and paramagnetically strong bimodal chitosan nanoparticles (BCNPs). The W/O microemulsion system provides a confined environment for producing monodispersed BCNPs. Average particle size as estimated by the Transmission Electron Microscopy was 28 nm. The water to surfactant molar ratio of 22 produced small size fairly monodispersed BCNPs. Fluorescein isothiocyanate (FITC, a fluorescent dye) and Gd-DOTA (a paramagnetic Gd ion chelating agent) were covalently attached to chitosan polymer backbone prior to BCNP synthesis. The purpose of the covalent attachment of fluorescent and paramagnetic labels to chitosan is to prevent leakage of these labels from the BCNPs. The BCNPs were cross-linked with tartaric acid using water-soluble carbodiimide coupling chemistry in order to maintain particulate integrity. Zeta potential value of +27.6 mV confirmed positive surface charge of cross-linked BCNPs. Fluorescence excitation and emission spectra of BCNPs were similar to that of bare FITC spectra, showing characteristic 520 nm emission at the 490 excitation. Paramagnetic gadolinium ion (Gd3+) concentration in the BCNPs was determined by inductively coupled plasma (ICP) emission spectroscopy. The longitudinal (T1) and transverse (T2) proton relaxation times were determined as a function of Gd3+ concentration in the BCNPs at 4.7 Tesla. Proton relaxivity (R1 value) of BCNPs was calculated to be 41.1 mM Gd(-1)s(-1). The reported R1 value of Gd-DOTA chelates is however 5.8 mM Gd(-1)s(-1). High proton relaxivity of BCNPs is attributed to hydrated chitosan environment around Gd chelates which additionally contributed to overall water exchange process. To demonstrate in vitro bioimaging capability, J774 macrophage cells were incubated with BCNPs. Confocal images clearly showed BCNP uptake by J774 cells. Internalization of BCNPs was confirmed by co-labeling J774 cells with a red-emitting membrane dye. BCNP green emission was mostly observed from middle of cells and within the red-emitting membrane boundary.

T2 exchange agents: A new class of paramagnetic MRI contrast agent that shortens water T2 by chemical exchange rather than relaxation

Exchange of water molecules between the frequency-shifted inner-sphere of a paramagnetic lanthanide ion and aqueous solvent can shorten the T(2) of bulk water protons. The magnitude of the line-broadening T(2) exchange (T(2exch)) is determined by the lanthanide concentration, the chemical shift of the exchanging water molecule, and the rate of water exchange between the two pools. A large T(2exch) contribution to the water linewidth was initially observed in experiments involving Eu(3+)-based paramagnetic chemical exchange saturation transfer agents in vivo at 9.4 T. Further in vitro and in vivo experiments using six different Eu(3+) complexes having water exchange rates ranging from zero (no exchange) to 5 × 10(6) s(-1) (fast exchange) were performed. The results showed that the exchange relaxivity (r(2exch)) is small for complexes having either very fast or very slow exchange, but reaches a well-defined maximum for complexes with intermediate water exchange rates. These experimental results were verified by Bloch simulations for two site exchange. This new class of T(2exch) agent could prove useful in the design of responsive MRI contrast agents for molecular imaging of biological processes.

Eu(III) complexes as anion-responsive luminescent sensors and paramagnetic chemical exchange saturation transfer agents.

The Eu(III) complex of (1S,4S,7S,10S)-1,4,7,10-tetrakis(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane (S-THP) is studied as a sensor for biologically relevant anions. Anion interactions produce changes in the luminescence emission spectrum of the Eu(III) complex, in the (1)H NMR spectrum, and correspondingly, in the PARACEST spectrum of the complex (PARACEST = paramagnetic chemical exchange saturation transfer). Direct excitation spectroscopy and luminescence lifetime studies of Eu(S-THP) give information about the speciation and nature of anion interactions including carbonate, acetate, lactate, citrate, phosphate, and methylphosphate at pH 7.2. Data is consistent with the formation of both innersphere and outersphere complexes of Eu(S-THP) with acetate, lactate, and carbonate. These anions have weak dissociation constants that range from 19 to 38 mM. Citrate binding to Eu(S-THP) is predominantly innersphere with a dissociation constant of 17 μM. Luminescence emission peak changes upon addition of anion to Eu(S-THP) show that there are two distinct binding events for phosphate and methylphosphate with dissociation constants of 0.3 mM and 3.0 mM for phosphate and 0.6 mM and 9.8 mM for methyl phosphate. Eu(THPC) contains an appended carbostyril derivative as an antenna to sensitize Eu(III) luminescence. Eu(THPC) binds phosphate and citrate with dissociation constants that are 10-fold less than that of the Eu(S-THP) parent, suggesting that functionalization through a pendent group disrupts the anion binding site. Eu(S-THP) functions as an anion responsive PARACEST agent through exchange of the alcohol protons with bulk water. The alcohol proton resonances of Eu(S-THP) shift downfield in the presence of acetate, lactate, citrate, and methylphosphate, giving rise to distinct PARACEST peaks. In contrast, phosphate binds to Eu(S-THP) to suppress the PARACEST alcohol OH peak and carbonate does not markedly change the alcohol peak at 5 mM Eu(S-THP), 15 mM carbonate at pH 6.5 or 7.2. This work shows that the Eu(S-THP) complex has unique selectivity toward binding of biologically relevant anions and that anion binding results in changes in both the luminescence and the PARACEST spectra of the complex.

Dynamic nuclear polarization polarizer for sterile use intent

A novel polarizer based on the dissolution-dynamic nuclear polarization (DNP) method has been designed, built and tested. The polarizer differs from those previously described by being designed with sterile use intent and being compatible with clinical use. The main features are: (1) an integral, disposable fluid path containing all pharmaceuticals constituting a sterile barrier, (2) a closed-cycle cryogenic system designed to eliminate consumption of liquid cryogens and (3) multi-sample polarization to increase throughput. The fluid path consists of a vial with the agent to be polarized, a pair of concentric inlet and outlet tubes connected to a syringe with dissolution medium and a receiver, respectively. The fluid path can operate at up to 400 K and 2.0 MPa and generates volumes as high as 100 mL. An inline filter removes the amount of electron paramagnetic agent in the final product by more than 100-fold in the case of [1-(13)C]pyruvate. The system uses a sorption pump in conjunction with a conventional cryocooler. The system operates through cycles of pumping to low temperature and regeneration of the sorption pump. The magnet accommodates four samples at the same time. A temperature of less than 1 K was achieved for 68 h (no sample heat loads) with a liquid helium volume of 2.4 L. The regeneration of the liquid helium could be achieved in less than 10 h, and the transition to cold (< 1.2 K) was achieved in less than 90 min. A solid state polarization of 36 ± 4% for [1-(13)C]pyruvic acid was obtained with only 10 mW of microwave power. The loading of a sample adds less than 50 J of heat to the helium bath by introducing the sample over 15 min. The heat load imposed on the helium bath during dissolution was less than 70 J. The measured liquid state polarization was 18 ± 2%.

Europium(III) DOTA-Derivatives Having Ketone Donor Pendant Arms Display Dramatically Slower Water Exchange

A series of new 1,4,7,10-tetraazacyclododecane-derivatives having a combination of amide and ketone donor groups as side-arms were prepared, and their complexes with europium(III) studied in detail by high resolution NMR spectroscopy. The chemical shift of the Eu(3+)-bound water resonance, the chemical exchange saturation transfer (CEST) characteristics of the complexes, and the bound water residence lifetimes (τ(m)) were found to vary dramatically with the chemical structure of the side-arms. Substitution of ketone oxygen donor atoms for amide oxygen donor atoms resulted in an increase in residence water lifetimes (τ(m)) and a decrease in chemical shift of the Eu(3+)-bound water molecule (Δω). These experimental results along with density functional theory (DFT) calculations demonstrate that introduction of weakly donating oxygen atoms in these complexes results in a much weaker ligand field, more positive charge on the Eu(3+) ion, and an increased water residence lifetime as expected for a dissociative mechanism. These results provide new insights into the design of paramagnetic CEST agents with even slower water exchange kinetics that will make them more efficient for in vivo imaging applications.

Integrin Targeted MR Imaging

Magnetic resonance imaging (MRI) is a powerful medical diagnostic imaging modality for integrin targeted imaging, which uses the magnetic resonance of tissue water protons to display tissue anatomic structures with high spatial resolution. Contrast agents are often used in MRI to highlight specific regions of the body and make them easier to visualize. There are four main classes of MRI contrast agents based on their different contrast mechanisms, including T(1), T(2), chemical exchange saturation transfer (CEST) agents, and heteronuclear contrast agents. Integrins are an important family of heterodimeric transmembrane glycoproteins that function as mediators of cell-cell and cell-extracellular matrix interactions. The overexpressed integrins can be used as the molecular targets for designing suitable integrin targeted contrast agents for MR molecular imaging. Integrin targeted contrast agent includes a targeting agent specific to a target integrin, a paramagnetic agent and a linker connecting the targeting agent with the paramagnetic agent. Proper selection of targeting agents is critical for targeted MRI contrast agents to effectively bind to integrins for in vivo imaging. An ideal integrin targeted MR contrast agent should be non-toxic, provide strong contrast enhancement at the target sites and can be completely excreted from the body after MR imaging. An overview of integrin targeted MR contrast agents based on small molecular and macromolecular Gd(III) complexes, lipid nanoparticles and superparamagnetic nanoparticles is provided for MR molecular imaging. By using proper delivery systems for loading sufficient Gd(III) chelates or superparamagnetic nanoparticles, effective molecular imaging of integrins with MRI has been demonstrated in animal models.

Yeast cell wall particles: a promising class of nature-inspired microcarriers for multimodal imaging

This communication demonstrates that yeast cell wall particles (YCWPs) are a promising class of nature-inspired biocompatible microcarriers for the delivery of amphipathic/lipophilic imaging reporters. When a paramagnetic MRI agent is loaded, the longitudinal relaxivity per particle at 0.5 T is the highest ever reported for Gd-based systems.

Folate receptor-targeted fluorescent paramagnetic bimodal liposomes for tumor imaging

Rationale and objectiveReceptor-targeted delivery of imaging and therapeutic agents can lead to enhanced efficacy for both. Multimodality imaging offers unique advantages over traditional single modality imaging. Tumor marker folate receptor (FR)-targeted fluorescent paramagnetic bimodal liposomes were synthesized to co-deliver paramagnetic and fluorescence agents for magnetic resonance (MR) and optical bimodal imaging contrast enhancement.Materials and methodsFluorescent and paramagnetic bimodal liposomes were synthesized with a mean diameter of 136 nm and a low polydispersity index. The liposomes incorporated folate-PEG3350-CHEMS for FR targeting, Gd(III)[N,N-Bis-stearylamidomethyl-N’-amidomethyl]diethylenetriamine tetraacetic acid (Gd-DTPA-BSA) for MR contrast, and calcein for fluorescence. To determine the specificity and efficiency of delivery, the liposomes were evaluated in FR-positive KB and HeLa cells and FR-negative A549 cells, which were analyzed by fluorescence microscopy, magnetic resonance imaging (MRI), and flow cytometry (FCM).ResultsFR-specific and efficient cellular uptake of the FR-targeted bimodal liposomes was confirmed by fluorescence microscopy and by FCM. The mean fluorescence intensity (MFI) of KB cells treated with FR-targeted liposomes was 45× that of cells treated with nontargeted liposomes, and 18× that of cells treated with FR-targeted liposomes and excess folic acid (FA). The MFI of HeLa cells treated with targeted liposomes was 33× that of nontargeted liposomes, and was 16× that of the mixture of targeted liposomes and free FA. In contrast, the MFI of A549 cells treated with FR-targeted liposomes was nearly the same as those treated with nontargeted liposomes. The T1-weighted MR images of HeLa and KB cells incubated with FR-targeted liposomes had much higher signal intensity than those treated with nontargeted liposomes or free Gd-DTPA. Furthermore, the FR-targeting effect could be blocked by excess free FA.ConclusionFR-targeted fluorescent paramagnetic bimodal liposomes provided a novel platform for bimodal tumor imaging and theranostic delivery.

Paramagnetic relaxation of nuclear singlet states

Nuclear singlet states often display lifetimes that are much longer than conventional nuclear spin relaxation times. Here we investigate the effect of dissolved paramagnetic species on the singlet relaxation of proton pairs in solution. We find a linear correlation between the singlet relaxation rate constant T and the longitudinal relaxation rate constant T. The slope of the correlation depends on the nature of the paramagnetic relaxation agent, but typically, singlet states are between two to three times less sensitive to paramagnetic relaxation than conventional nuclear magnetization. These observations may be interpreted using a model of partially-correlated local fields acting on the nuclear sites. We explore the effect on singlet relaxation of adding a metal-ion-chelating agent to the solution. We also investigate the effect of ascorbate, which reacts with dissolved oxygen.

Screening of CEST MR contrast agents.

There has been a tremendous amount of interest in developing new MR contrast agents for cellular and molecular imaging applications such as the visualization of tumors, highlighting areas of angiogenesis, highlighting of contrast agent-labeled therapeutic stem cells, and highlighting of contrast agent-labeled drug delivery vehicles. The contrast properties of paramagnetic and super-paramagnetic relaxation-based agents have allowed MR imaging to be used as a tool for all of the above applications. However, a new class of MR contrast agents, chemical exchange saturation transfer (CEST) agents, provides additional features such as (1) the ability to highlight multiple biological events at once within an image through the distinguishability of the different CEST contrast agents, (2) the ability to toggle the contrast "off-to-on" by applying a saturation pulse, and (3) potentially providing more information about the environment surrounding the contrast agent such as the pH or concentration of metabolites. In this chapter, we will focus on the methods which can be used in terms of acquisition schemes and hardware to screen these agents through MR imaging.

Calcium-responsive paramagnetic CEST agents

The assessment of changes in the extracellular calcium concentration by magnetic resonance imaging would be a valuable biomedical research tool to monitor brain neuronal activity. In this perspective, we report here the synthesis of novel ligands consisting of tetraamide and bisamide derivatives of cyclen, L(1) and L(2), respectively, each bearing imino(diacetate) moieties for Ca(2+) binding. Yb(3+) and Eu(3+) complexes are investigated as chemical exchange saturation transfer (CEST) agents that respond to the presence of Ca(2+). A CEST effect is observed for both YbL(1) and EuL(1) complexes (B=11.7T), originating from the slow exchange of the amide protons and those of the coordinated water, respectively, whilst no CEST is detected for complexes of L(2). Upon calcium binding, the CEST effect decreases considerably (from 60% to 20% for YbL(1) and from 35% to 10% for EuL(1)). A similar variation is observed in the presence of Mg(2+). The affinity constants between the lanthanide complexes and the alkaline earth metal ions have been estimated from the variation of the CEST effect to be K(YbL(1)-Ca)(aff) = 8 ± 2M(-1), K(YbL(1)-Mg)(aff) = 23 ± 3M(-1) and K(EuL(1)-Ca)(aff) = 10 ± 3M(-1). These low values imply the coordination of the alkaline earth ions to a single iminodiacetate arm. Ca(2+)/Mg(2+) binding to the lanthanide complexes slows down the exchange of the amide protons on YbL(1) which is responsible for the diminished CEST effect. This has been evidenced by assessing the proton exchange rates from the dependency of the CEST effect on the saturation time and the saturation power, in the absence and in the presence of Ca(2+) and Mg(2+). The applicability of the PARACEST MRI agents for Ca(2+) detection has been evaluated on a 16T MRI scanner.

High‐throughput screening of chemical exchange saturation transfer MR contrast agents

A new high-throughput MRI method for screening chemical exchange saturation transfer (CEST) agents is demonstrated, allowing simultaneous testing of multiple samples with minimal attention to sample configuration and shimming of the main magnetic field (B(0)). This approach, which is applicable to diamagnetic, paramagnetic and liposome CEST agents, employs a set of inexpensive glass or plastic capillary tubes containing CEST agents put together in a cheap plastic tube holder, without the need for liquid between the tubes to reduce magnetic susceptibility effects. In this setup, a reference image of direct water saturation spectra is acquired in order to map the absolute water frequency for each volume element (voxel) in the sample image, followed by an image of saturation transfer spectra to determine the CEST properties. Even though the field over the total sample is very inhomogeneous due to air-tube interfaces, the shape of the direct saturation spectra is not affected, allowing removal of susceptibility shift effects from the CEST data by using the absolute water frequencies from the reference map. As a result, quantitative information such as the mean CEST intensity for each sample can be extracted for multiple CEST agents at once. As an initial application, we demonstrate rapid screening of a library of 16 polypeptides for their CEST properties, but in principle the number of tubes is limited only by the available signal-noise-ratio, field of view and gradient strength for imaging.

A concentration-independent method to measure exchange rates in PARACEST agents

The efficiency of chemical exchange dependent saturation transfer (CEST) agents is largely determined by their water or proton exchange kinetics, yet methods to measure such exchange rates are variable and many are not applicable to in vivo measurements. In this work, the water exchange kinetics of two prototype paramagnetic agents (PARACEST) are compared by using data from classic NMR line-width measurements, by fitting CEST spectra to the Bloch equations modified for chemical exchange, and by a method where CEST intensity is measured as a function of applied amplitude of radiofrequency field. A relationship is derived that provides the water exchange rate from the X-intercept of a plot of steady-state CEST intensity divided by reduction in signal caused by CEST irradiation versus 1/omega(1)(2), referred to here as an omega plot. Furthermore, it is shown that this relationship is independent of agent concentration. Exchange rates derived from omega plots using either high-resolution CEST NMR data or CEST data obtained by imaging agree favorably with exchange rates measured by the more commonly used Bloch fitting and line-width methods. Thus, this new method potentially allows access to a direct measure of exchange rates in vivo, where the agent concentration is typically unknown.

Ischemic hyperintensities on T1-weighted magnetic resonance imaging of patients with stroke: New insights from susceptibility weighted imaging

Hyperintensities on T1-weighted magnetic resonance imaging (MRI) in the setting of brain ischemia are usually considered hemorrhagic transformations. Such changes can also be seen due to "incomplete infarction" with selective neuronal loss. Arguments regarding the cause of these T1 hyperintensities have shuttled between gemistocytic astrocyte accumulation, tissue calcification and paramagnetic substance deposition. Susceptibility weighted imaging (SWI), a sensitive modality for detecting paramagnetic agents and blood products, has never been used to resolve this issue. The study was aimed to evaluate the SWI signal changes of T1 hyperintense lesion in stroke patients and understand its usefulness in differentiating a hemorrhagic infarct and an incomplete infarct. All the seven patients with infarct, having hyperintensities on T1 weighted MR imaging seen over the last one year were subjected to SWI. In none of the patients SWI failed to show any blooming. By doing SWI for T1-weighted hyperintensities, we can differentiate hemorrhagic infarct and a non-hemorrhagic "incomplete infarct". This differentiation will immensely help in planning management strategy and prognostication.

SEMPRE: Spectral Editing Mediated by Paramagnetic Relaxation Enhancement

Paramagnetic relaxation enhancement (PRE) has become a useful and widely applied tool in biomolecular NMR spectroscopy. In particular investigations of large complexes or transient contacts benefit from PRE effects. Frequently such studies involve modification of the biomacromolecules under study. We here present a method for editing NMR spectra by utilizing a soluble gadolinium complex that broadens nuclear spins being at or close to the macromolecule-solvent interface. NOE signals in NOESY spectra are selectively attenuated if surface exposed nuclear spins are involved. HSQC-type spectra with paramagnetic agent contain only signals of the interior of the protein, while the corresponding difference spectra harbor signals allocated to surface spins. Thus, the number of signals can be reduced helping to minimize spectral overlap in large proteins. The method reveals additional information about the localization of spins being helpful for structure determination of large complexes.

Non-PEGylated liposomes for convection-enhanced delivery of topotecan and gadodiamide in malignant glioma: initial experience

Convection-enhanced delivery (CED) of highly stable PEGylated liposomes encapsulating chemotherapeutic drugs has previously been effective against malignant glioma xenografts. We have developed a novel, convectable non-PEGylated liposomal formulation that can be used to encapsulate both the topoisomerase I inhibitor topotecan (topoCED™) and paramagnetic gadodiamide (gadoCED™), providing an ideal basis for real-time monitoring of drug distribution. Tissue retention of topoCED following single CED administration was significantly improved relative to free topotecan. At a dose of 10 μg (0.5 mg/ml), topoCED had a half-life in brain of approximately 1 day and increased the area under the concentration–time curve (AUC) by 28-fold over free topotecan (153.8 vs. 5.5 μg day/g). The combination of topoCED and gadoCED was found to co-convect well in both naïve rat brain and malignant glioma xenografts (correlation coefficients 0.97–0.99). In a U87MG cell assay, the 50% inhibitory concentration (IC50) of topoCED was approximately 0.8 μM at 48 and 72 h; its concentration–time curves were similar to free topotecan and unaffected by gadoCED. In a U87MG intracranial rat xenograft model, a two-dose CED regimen of topoCED co-infused with gadoCED greatly increased median overall survival at dose levels of 0.5 mg/ml (29.5 days) and 1.0 mg/ml (33.0 days) vs. control (20.0 days; P < 0.0001 for both comparisons). TopoCED at higher concentrations (1.6 mg/ml) co-infused with gadoCED showed no evidence of histopathological changes attributable to either agent. The positive results of tissue pharmacokinetics, co-convection, cytotoxicity, efficacy, and lack of toxicity of topoCED in a clinically meaningful dose range, combined with an ideal matched-liposome paramagnetic agent, gadoCED, implicates further clinical applications of this therapy in the treatment of malignant glioma.