Study of proton spin-lattice relaxation variation induced by paramagnetic antibodies

A murine anti-human melanoma monoclonal antibody fragment was labeled with gadolinium and its proton relaxation efficiency compared to controls at frequencies ranging from 2 to 300 MHz. Relaxation time variations were about 30-40% in 10-15 microM solutions. The labeled fragment showed proton relaxation enhancement relative to free gadolinium, while preserving its immunoreactivity. A tentative labeling of a melanoma pellet by means of the fragment, just at the borderline of a minimum expected T1 variation, gave no detectable difference.     

Insights on the relaxation of liposomes encapsulating paramagnetic Ln‐based complexes

Purpose: To describe and quantify the different relaxation mechanisms operating in suspensions of liposomes that encapsulate paramagnetic lanthanide(III) complexes. Theory and Methods: The transverse relaxation rate of lanthanide‐loaded liposomes receives contribution from the exchange between intraliposomal and bulk water protons, and from magnetic susceptibility effects. Phospholipids vesicles encapsulating different Ln(III)‐HPDO3A complexes (Ln = Eu, Gd, or Dy) were prepared using the conventional thin film rehydration method. Relaxation times (T₁, T₂, and T₂^*) were measured at 14 Tesla (T) and 25°C. The effect of compartmentalization of the paramagnetic agent inside the liposomal cavity was evaluated by means of an IRON‐modified MRI sequence. Results: NMR measurements demonstrated that Curie spin relaxation is the dominant contribution (> 90%) to the observed transverse relaxation rate of paramagnetic liposomes. This was further confirmed by MRI that showed the ability of the liposome entrapped lanthanide complexes to generate IRON‐MRI positive contrast in a size dependent manner. Conclusion: The Curie spin relaxation mechanism is by far the principal mechanism involved in the T₂ shortening of the water protons in suspension of paramagnetic liposomes at 14T. The access to IRON contrast extends the potential of such nanosystems as MRI contrast agents. Magn Reson Med 74:468–473, 2015. © 2014 Wiley Periodicals, Inc.     

Evidence for a contribution of paramagnetic ions to water proton spin-lattice relaxation in normal and malignant mouse tissues.

Paramagnetic ions complexed to proteins may lose, retain, or enhance solvent paramagnetic relaxation (SPR) relative to free solution. We measured T1 and T2 of three mouse cancers, their normal counterparts, and six additional tissues. Long T1 of cancers was not caused by necrosis or by different contents of water, fat, or blood. Dissociable (TCA-extractable) and nondissociable (ashed) Mn, Cu, and Fe were measured by AA. Cancers had less Mn, Cu, and Fe than did normal counterparts. All 12 tissues had inverse correlations between T1 and dissociable Mn and Cu. For Mn alone to account for reduced T1, the extent to which SPR of the Mn-protein complexes would be enhanced is by factors of 0.6 to 13, below the maximum observed in Mn-enzymes. Different amounts of paramagnetic ion-protein complexes may account for part of the differences in T1 of water protons in different tissues, and the longer T1 of cancer cell water may be caused in part by reduced amounts of such complexes.     

Influence of paramagnetic ions and pH on proton NMR relaxation of biologic fluids

The extent to which various concentrations of the paramagnetic metal ions [gadolinium (III), manganese (II), chromium (III), iron (III), nickel (II), copper (II), and cobalt (II)] affect proton magnetic relaxation times of distilled water, 4% human serum albumin (HSA), and dog plasma was studied in vitro. The pH of water and HSA varied from 4 to 8. Nuclear magnetic resonance relaxation parameters, T1 and T2, were measured at 10.7 MHz using inversion recovery and spin-echo radiofrequency sequences, respectively. The presence of Mn(II), Gd(III) and Cr(III) in water significantly reduced T1, while Fe(III), Ni(II), Cu(II) and Co(II) had only a minimal effect. In 4% HSA and dog plasma Mn(II) and Cu(II) had the greatest effect on T1. At neutral pH, Gd(III) and Cr(III) had little effect on T1, while Mn(II) induced a large shortening of T1. All of the metal ions changed T2 less than T1. These differences in proton relaxation enhancement caused by the various ions in the three solutions studied are due to variations in the effective magnetic moment, the degree of binding of the ions to protein, and the chemical form of the ion associated with changes in pH. Thus, it is impossible to predict the effect of metal ions on proton relaxation in vivo based solely on in vitro studies, because of the complexity of various biologic fluids in vivo.     

Gadolinium-labeled liposomes containing paramagnetic amphipathic agents: targeted MRI contrast agents for the liver

Unique paramagnetic liposomal contrast agents were synthesized and utilized for selective augmentation of T1 MR imaging of the livers of normal Balb/c mice. Amphipathic gadolinium complexes, which mimic phospholipids, were incorporated into the lamella of small unilamellar liposomes (SUV) such that they became an integral part of its surface. T1 signal enhancement of normal liver approached 150% after iv administration of the paramagnetic liposomes, determined by experiments performed on a 1.9-T, experimental whole-body MRI unit. Tracer studies utilizing gadolinium-153-tagged SUV revealed that the agents exhibited excellent in vivo stability, compared to liposomal preparations in which paramagnetic agents are simply entrapped in the aqueous core of the liposome vesicle.     

The magnetic field dependence of transverse water proton relaxation induced by nitroxide radicals

The magnetic field dependence of the water proton T2 is calculated for aqueous solutions of nitroxides based on a detailed analysis of early T1 measurements on nitroxide solutions made as a function of magnetic field. The results parallel those for T1 closely and, unlike metal systems, the implications for magnetic imaging are similar.     

Multiexponential proton relaxation processes of compartmentalized water in gels.

The proton relaxation times, T1 and T2, of water in Sephadex gels, exhibiting pores of varying size (i.e., with exclusion limits of molecular weight between 10(3) and 10(5)) and water contents in the range 30 to 70% (w/w, weight of water to total weight), were measured at 20 MHz in the temperature range 5 to 50 degrees C. Multiexponential analysis of the relaxation curves revealed the existence of two relaxation components in all gel systems. A component with long T1 and T2 (T1,1 and T2,1) is associated with a large water fraction alpha 1,1 and alpha 2,1 and a component with short T1 and T2 (T1,2 and T2,2) with a small water fraction alpha 1,2 and alpha 2,2. An analysis of the temperature behavior of the relaxation components gives insight into the relaxation mechanisms. The relaxation process in water, compartmentalized in the gel matrix, is mainly controlled by dipole-dipole interactions. In addition, proton exchange processes between hydration water and hydroxyl groups of the matrix chain contribute under specific conditions and lead to a dramatic enhancement of the relaxation rate. In particular, for gels with small pores and with low water content proton exchange is observed. Compartments of water in gels could be models for compartments of water in biological tissues.     

Molecular basis of water proton relaxation in gels and tissue.

An extensive set of water-1H magnetic relaxation dispersion (MRD) data are presented for aqueous agarose and gelatin gels. It is demonstrated that the EMOR model, which was developed in a companion paper to this study (see Halle, this issue), accounts for the dependence of the water-1H spin-lattice relaxation rate on resonance frequency over more than four decades and on pH. The parameter values deduced from analysis of the 1H MRD data are consistent with values derived from 2H MRD profiles from the same gels and with small-molecule reference data. This agreement indicates that the water-1H relaxation dispersion in aqueous biopolymer gels is produced directly by exchange-mediated orientational randomization of internal water molecules or labile biopolymer protons, with little or no role played by collective biopolymer vibrations or coherent spin diffusion. This ubiquitous mechanism is proposed to be the principal source of water-1H spin-lattice relaxation at low magnetic fields in all aqueous systems with rotationally immobile biopolymers, including biological tissue. The same mechanism also contributes to transverse and rotating-frame relaxation and magnetization transfer at high fields.     

Magnetic resonance imaging of rabbit brain after intracarotid injection of large multivesicular liposomes containing paramagnetic metals and DTPA

A procedure has been developed for the preparation of large (10- to 80-microns-diameter) multivesicular liposomes that contain magnetic resonance contrast agent (DTPA and either manganese or gadolinium). Blue dextran was observed to induce the formation of the large liposomes with dioleoylphosphatidylcholine and cholesterol (1:1 molar ratio) and with dipalmitoylphosphatidylcholine and cholesterol (1:1 molar ratio). The formation of the large liposomes is dependent upon mixing the blue dextran with the lipid films at temperatures above the transition point of the lipids. Tracer amounts of 153Gd were added to the aqueous phase to permit quantitation of the recovery of encapsulated materials. Liposomes that were prepared using equimolar ratios of phospholipid and cholesterol were stable in serum for more than 12 h. The ultrastructure of the large multivesicular liposomes reveals the existence of individual vesicles (greater than 2 micron diameter) bound together by a multilamellar coating. When injected into the internal carotid artery of the rabbit, the large liposomes became entrapped in the vascular bed primarily in the frontal and occipital regions of brain. The resulting emboli may provide a means to deliver drugs to a specific site in brain, such as a tumor, if the vascular bed of the site can be cannulated precisely.     

Persistent contrast enhancement by sterically stabilized paramagnetic liposomes in murine melanoma.

In the present research, we investigated the use of paramagnetic liposomes as contrast agents (CAs) for the detection of solid tumors. The liposomes were sterically stabilized by a polyethylene glycol (PEG) coating, and their size was constrained to approximately 100 nm. Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-diethylene-triaminepentaacetate (DMPE-DTPA) was used as the gadolinium-carrying fatty acid chain. The relaxation properties were characterized through nuclear magnetic relaxation dispersion (NMRD) measurements, and analyzed with the use of theories and computer programs that are adequate for slowly rotating systems. Their relaxivity at 1.5 T was found to be acceptable for in vivo use. We then tested the liposomes against B16-F10 murine melanomas using standard T1-weighted schemes at 1.5 T, and concentrations corresponding to 0.03 mmol/kg of gadolinium (i.e., three to six times lower than the concentration of the small gadolinium complexes in clinical use). The blood half-life was found to be 120 +/- 20 min. The experiments show a good contrast enhancement in the tumor (33% +/- 22%) 2 hr after administration, a further increase (43 +/- 27%) 20 hr after administration, and a decrease (25% +/- 14%) 54 hr after administration. High persistence of the CA was also observed in the liver and intestine, as expected in a hepatobiliar excretion pathway.     

Measurement of solute proton spin-lattice relaxation times in water using the 1,3,3,1 sequence

1H NMR spin-lattice relaxation times (T1) of the N-CH3 proton resonances of phosphocreatine (PCr) and creatine (Cr) in water solutions were obtained using the 1,3,3,1 pulse sequence. These T1 values were equivalent to those obtained in D2O and water using either the conventional inversion-recovery experiment or the 1,3,3,1 pulse sequence. Thus, the 1,3,3,1 sequence of proton NMR can provide an independent means along with phosphorous NMR for assess PCr and for the study of the creatine kinase reaction (PCr + ADP in equilibrium ATP + Cr) in aqueous solutions and perhaps in biological preparations.     

Magnetization transfer contrast (MTC) and tissue water proton relaxation in vivo

In this study the exchange between 1H magnetization in "free" water (1Hf) and that in a pool with restricted motion (1Hr) was observed in tissues in vivo using NMR saturation transfer methods. Exchange between these two pools was demonstrated by a decrease in the steady-state magnetization and relaxation times of 1Hf with radiofrequency irradiation of 1Hr. The pseudo-first-order rate constant for the movement of magnetization from 1Hf to 1Hr was approximately 1 s-1 in kidney and approximately 3 s-1 in skeletal muscle in vivo. Proton NMR imaging demonstrated that this exchange was tissue specific and generated a novel form of NMR image contrast. The extent of exchange between 1Hf and 1Hr as well as the topological correlation of the exchange with relaxation weighted images suggests that this pathway is a major determinant of the observed relaxation properties of water 1H in vivo.     

Properties of liposomes containing 212Pb

The reverse phase evaporation method was used to prepare lipid bilayer membrane vesicles containing 212Pb and other markers of high specific activity. Electron microscopy and microfiltration were used to measure the sizes of the liposomes. Isotopes were released from the liposomes during exposure to serum and this leakage was prevented by complexing of small molecules with proteins or by precipitating particulate complexes within the liposomes. The in vivo distribution of 212Pb liposomes differed from the distribution of free 212Pb in that the reticuloendothelial system cleared the liposomes. Liposomes with surface dinitrophenol hapten were highly immunogenic and the humoral response to dinitrophenol was nonspecifically suppressed by 212Pb liposomes.     

Longitudinal proton relaxation rates in rabbit tissues after intravenous injection of free and chelated Mn2+

The factors that determine the field-dependent increase in 1/T1 of tissue water protons were investigated for MnCl2 and Mn2+ (PDTA) (1,3-propylenediamine-N,N',N',N'-tetraacetic acid) introduced intravenously into rabbits. Mn2+ was used in preference to other paramagnetic ions in part because of the distinct NMRD profiles (magnetic field dependence of 1/T1) of free Mn2+ ions, their small chelate complexes, and their macromolecular conjugates, and in part because the relatively low toxicity of Mn2+ is favorable for animal studies. Tissue content of Mn2+ was determined in all samples by inductively coupled plasma analyses the state of Mn2+ in excised tissues was determined from the form of the 1/T1 NMRD profile of water protons; and distribution of contrast agent within tissue and access of water on a T1 time scale were determined by double-exponential analyses of proton relaxation behavior in intact doped tissue, as well as by the change of single-exponential relaxation rates and proton signal intensity upon gentle disruption of the tissue. MnCl2 is found in all tissues, except fat and skeletal muscle, but liver is most avid at low dose, and Mn2+ accumulates in spleen after high doses. Chelation targets Mn2+ to liver and kidney, saturating the liver chemically at relatively low dose. We suggest that pronounced increase in tissue relaxivity results from irrotationally bound Mn2+, ostensibly associated with the polar head groups of cell membranes. Compartmentalization of contrast agent and restricted diffusion of tissue water influences the maximum relaxation rates attainable, so that there is an optimal dose of these contrast agents which is rather low.     

Quantitative brain proton MR spectroscopy based on measurement of the relaxation time T1 of water

PURPOSE: To provide a straightforward method for metabolite quantitation in the brain. Tissue water concentration can be determined in a voxel by measuring T(1) and it may provide an internal reference for the calculation of the metabolite concentrations. MATERIALS AND METHODS: Water-suppressed stimulated echo acquisition mode spectra were obtained at 1.5T, and the tissue water content was calculated from T(1). RESULTS: The calculated water content values demonstrated very good agreement with literature data. Metabolite concentrations (mmol/liter) in the gray and white matter: N-acetyl-aspartate = 14.02 +/- 1.93, creatine = 9.98 +/- 1.03, and choline = 1.14 +/- 0.24; N-acetyl-aspartate = 11.08 +/- 2.24, creatine = 7.83 +/- 0.66, and choline = 2.05 +/- 0.38, respectively. CONCLUSION: The water content calculated from T(1) can yield an internal reference in MR spectroscopy, and the accurate measurement of metabolite concentrations is feasible. The proposed method is simple and can readily be applied in any MR center without the need for complicated corrections or calibration procedures.     

Effects of spatial distribution on proton relaxation enhancement by particulate iron oxide

The proton relaxation effect of superparamagnetic iron oxide (SPIO) particles under varying conditions of spatial distribution was investigated with use of phantoms. Agar phantoms containing various concentrations of SPIO or gadopentetate dimeglumine, with and without Sephadex beads, were studied. Phantoms with Sephadex had a heterogeneous spatial distribution of iron oxide, comparable to liver tissue in vivo. Relaxometry at 0.47 T showed decreased T2 relaxivity of SPIO in Sephadex phantoms compared with that in agar phantoms without Sephadex. On T2-weighted images obtained at 1.5 T, the signal intensity of Sephadex phantoms showed less SPIO relaxation effect than that of plain agar phantoms. Unlike SPIO, gadopentetate dimeglumine showed the same relaxivities and signal intensity in plain agar and Sephadex phantoms. The results show that the T2 relaxation effect of iron oxide depends on its spatial distribution. A heterogeneous spatial distribution, as in intact liver tissue, diminishes the T2 relaxivity of iron oxide particles.     

Effect of induced field inhomogeneity on transverse proton NMR relaxation in tissue water and model systems

The effect of induced field inhomogeneity (IFI) on transverse NMR relaxation of water protons in tissue has been investigated by examining the field dependence of the effective transverse relaxation rates (1/T2 eff) for in vitro canine brain tissue samples. At fields of 0.47, 2.35, 7.05 T (corresponding to 20, 100, and 300 MHz, respectively) the transverse relaxation rates for both white and gray matter samples follow a field dependence of the form 1/T2 eff = C0 + C1 B0, where B0 is the applied field. The linearly dependent term, C1 B0, which reflects the IFI contribution, does not contribute much (i.e., less than 20%) at fields less than 2.0 T. However, at greater field strengths the contribution is appreciable, e.g., greater than 60% at 7.0 T. Results from model systems of glass beads are also reported to illustrate IFI effects. For both the model systems and canine brain tissue samples, the effects of restricted diffusion are qualitatively evident in Hahn spin-echo experiments.     

High-resolution NMR imaging of paramagnetic liposomes targeted to a functionalized surface.

A major application of molecular MR imaging is receptor mapping of cells lining blood vessels with targeted contrast agents. Since these agents accumulate at interfaces, knowledge of their influence on the relaxation process in this specific configuration is a prerequisite for understanding their working principle. A methodology is presented to study the influence of targeted contrast agents on surface relaxation in vitro. Paramagnetic liposomes attached to a functionalized surface were studied with high-resolution NMR imaging. The surface was prepared by covering a solid substrate with a layer of collagen. Paramagnetic liposomes were targeted to this surface by functionalizing the liposomes with collagen adhesion protein CNA-35. With a saturation-recovery sequence, 1D magnetization profiles with a resolution of 5 microm were measured in water in contact with the surface. Analytical predictions, obtained with the Bloch-Torrey equation, perfectly agreed with the experimental data. Therefore, the magnitude of the surface relaxation rate could be determined from the measurements without any assumption. By using the relaxivity of liposome solutions the surface coverage by liposomes could be estimated. With the presented methodology the behavior of Gd-based targeted contrast agents at biological interfaces can be studied in vitro. Their influence on relaxation processes can be characterized and quantified.