We report the nanoscale loading and confinement of aquated Gd3+n-ion clusters within ultra-short single-walled carbon nanotubes (US-tubes); these Gd3+n@US-tube species are linear superparamagnetic molecular magnets with Magnetic Resonance Imaging (MRI) efficacies 40 to 90 times larger than any Gd3+-based contrast agent (CA) in current clinical use.

On the basis of structural considerations in the inner sphere of nine-coordinate, monohydrated Gd(III) poly(aminocarboxylate) complexes, we succeeded in accelerating the water exchange by inducing steric compression around the water binding site. We modified the common DTPA(5-) ligand (DTPA=(diethylenetriamine-N,N,N',N",N"-pentaacetic acid) by replacing one (EPTPA(5-)) or two (DPTPA(5-)) ethylene bridges of the backbone by propylene bridges, or one coordinating acetate by a propionate arm (DTTA-prop(5-)). The ligand EPTPA(5-) was additionally functionalized with a nitrobenzyl linker group (EPTPA-bz-NO(2) (5-)) to allow for coupling of the chelate to macromolecules. The water exchange rate, determined from a combined variable-temperature (17)O NMR and EPR study, is two orders of magnitude higher on [Gd(eptpa-bz-NO(2))(H(2)O)](2-) and [Gd(eptpa)(H(2)O)](2-) than on [Gd(dtpa)(H(2)O)](2-) (k(ex)298=150x10(6), 330x10(6), and 3.3x10(6) s(-1), respectively). This is optimal for attaining maximum proton relaxivities for Gd(III)-based, macrocyclic MRI contrast agents. The activation volume of the water exchange, measured by variable-pressure (17)O NMR spectroscopy, evidences a dissociative interchange mechanism for [Gd(eptpa)(H(2)O)](2-) (DeltaV(not equal sign)=(+6.6+/-1.0) cm(3) mol(-1)). In contrast to [Gd(eptpa)(H(2)O)](2-), an interchange mechanism is proved for the macrocyclic [Gd(trita)(H(2)O)](-) (DeltaV (not equal sign)=(-1.5+/-1.0) cm(3) mol(-1)), which has one more CH(2) group in the macrocycle than the commercial MRI contrast agent [Gd(dota)(H(2)O)](-), and for which the elongation of the amine backbone also resulted in a remarkably fast water exchange. When one acetate of DTPA(5-) is substituted by a propionate, the water exchange rate on the Gd(III) complex increases by a factor of 10 (k(ex)298=31x10(6) s(-1)). The [Gd(dptpa)](2-) chelate has no inner-sphere water molecule. The protonation constants of the EPTPA-bz-NO(2) (5-) and DPTPA(5-) ligands and the stability constants of their complexes with Gd(III), Zn(II), Cu(II) and Ca(II) were determined by pH potentiometry. Although the thermodynamic stability of [Gd(eptpa-bz-NO(2))(H(2)O)](2-) is reduced to a slight extent in comparison with [Gd(dtpa)(H(2)O)](2-), it is stable enough to be used in medical diagnostics as an MRI contrast agent. Therefore both this chelate and [Gd(trita)(H(2)O)](-) are potential building blocks for the development of high-relaxivity macromolecular agents.

A combined proton relaxivity and dynamic light scattering study has shown that aggregates formed in aqueous solution of water-soluble gadofullerenes can be disrupted by addition of salts. The salt content of fullerene-based materials will strongly influence properties related to aggregation phenomena, therefore their behavior in biological or medical applications. In particular, the relaxivity of gadofullerenes decreases dramatically with phosphate addition. Moreover, real biological fluids present a rather high salt concentration which will have consequences on fullerene aggregation and influence fullerene-based drug delivery.Water-soluble fullerene derivatives possess potential for biomedical applications as antioxidants, 1 anti-HIV drugs, 2 X-ray contrast agents, 3 bone-disorder drugs 4,5 and photosensitizers for photodynamic therapy. 6 In addition, endohedral metallofullerenes (M@C 2n ) have been suggested as nuclear medicines (M = Ho 3+ ) 7,8 , fluorescent tracers (M = Er 3+ ) 9 and MRI contrast agents (M = Gd 3+ ) 10-13 largely because the closed fullerene cage insures against toxic metal-ion release in vivo. Water-soluble members of the Gd@C 60 family The proton relaxivity, r 1 , which is the gauge of contrast agent efficiency, is remarkably higher (up to 10 times) for gadofullerenes than for typical clinical agents (r 1 is the paramagnetic longitudinal relaxation rate enhancement of water protons, referred to 1 mM concentration). 10-13 The electronic structure of Gd@C 60 involves the transfer of three electrons from the Gd atom to the cage resulting in seven unpaired electrons on the Gd 3+ center and one unpaired electron on the cage. The large relaxivity of the gadofullerenes has been attributed to their slow tumbling in solution and to the large number of surrounding water molecules. 13 This slow tumbling/rotation is related to aggregation phenomena in aqueous solution, and recently, in a variable-pH proton relaxation and dynamic light scattering (DLS) study, we confirmed a pHdependent aggregation of the gadofullerenes and proposed them as pH-responsive MRI contrast agents. 13With the aim of assessing the interaction between the aggregated gadofullerenes, Relaxivity is an ideal reporter of aggregation phenomena in paramagnetic solutions, as previously demonstrated in micellization of amphiphilic Gd 3+ chelates. 19 Disaggregation of the gadofullerenes leads to smaller and more rapidly tumbling entities, which will directly translate into lower relaxivities. On increasing PBS concentration in a gadofullerene solution, the relaxivity, indeed, decreases dramatically, indicating aggregate disruption (Figure 1)In order to separate the disaggregating effect of phosphate and sodium chloride in PBS, we have performed a relaxometric and DLS study of gadofullerene solutions at variable NaCl concentration (no phosphate). As Figure 2 shows, the relaxivity decrease on NaCl addition is also accompanied by a decrease of the hydrodynamic diameter, D H , thus confirming disaggregation as the most likely reason f...

The EPTPA5) chelate, which ensures fast water exchange in GdIII complexes, has been coupled to three different generations (5, 7, and 9) of polyamidoamine (PAMAM) dendrimers through benzylthiourea linkages (H5EPTPA = ethylenepropylenetriamine-N,N,N',N'',N''-pentaacetic acid). The proton relaxivities measured at pH 7.4 for the dendrimer complexes G5-(GdEPTPA)111, G7-(GdEPTPA)253 and G9-(GdEPTPA)1157 decrease with increasing temperature, indicating that, for the first time for dendrimers, slow water exchange does not limit relaxivity. At a given field and temperature, the relaxivity increases from G5 to G7, and then slightly decreases for G9 (r1 = 20.5, 28.3 and 27.9 mM(-1) s(-1), respectively, at 37 degrees C, 30 MHz). The relaxivities show a strong and reversible pH dependency for all three dendrimer complexes. This originates from the pH-dependent rotational dynamics of the dendrimer skeleton, which was evidenced by a combined variable-temperature and multiple-field 17O NMR and 1H relaxivity study performed at pH 6.0 and 9.9 on G5-(GdEPTPA)111. The longitudinal 17O and 1H relaxation rates of the dendrimeric complex are strongly pH-dependent, whereas they are not for the [Gd(EPTPA)(H2O)]2- monomer chelate. The longitudinal 17O and 1H relaxation rates have been analysed by the Lipari-Szabo spectral density functions and correlation times have been calculated for the global motion of the entire macromolecule (tau(gO)) and the local motion of the GdIII chelates on the surface (tau(lO)), correlated by means of an order parameter S2. The dendrimer complex G5-(GdEPTPA)111 has a considerably higher tau(gO) under acidic than under basic conditions (tau(298)gO = 4040 ps and 2950 ps, respectively), while local motions are less influenced by pH (tau(298)lO = 150 and 125 ps). The order parameter, characterizing the rigidity of the macromolecule, is also higher at pH 6.0 than at pH 9.9 (S2 = 0.43 vs 0.36, respectively). The pH dependence of the global correlation time can be related to the protonation of the tertiary amine groups in the PAMAM skeleton, which leads to an expanded and more rigid dendrimeric structure at lower pH. The increase of tau(gO) with decreasing pH is responsible for the pH dependent proton relaxivities. The water exchange rate on G5-(GdEPTPA)111(k(298)ex = 150 x 10(6) s(-1)) shows no significant pH dependency and is similar to the one measured for the monomer [Gd(EPTPA)(H2O)]2-. The proton relaxivity of G5-(GdEPTPA)111 is mainly limited by the important flexibility of the dendrimer structure, and to a small extent, by a faster than optimal water exchange rate.

With their nanoscalar, superparamagnetic Gd(3+)-ion clusters (1 x 5 nm) confined within ultrashort (20-80 nm) single-walled carbon nanotube capsules, gadonanotubes are high-performance T1-weighted contrast agents for magnetic resonance imaging (MRI). At 1.5 T, 37 degrees C, and pH 6.5, the r1 relaxivity (ca. 180 mM(-1) s(-1) per Gd(3+) ion) of gadonanotubes is 40 times greater than any current Gd(3+) ion-based clinical agent. Herein, we report that gadonanotubes are also ultrasensitive pH-smart probes with their r1/pH response from pH 7.0-7.4 being an order of magnitude greater than for any other MR contrast agent. This result suggests that gadonanotubes might be excellent candidates for the development of clinical agents for the early detection of cancer where the extracellular pH of tumors can drop to pH=7 or below. In the present study, gadonanotubes have also been shown to maintain their integrity when challenged ex vivo by phosphate-buffered saline solution, serum, heat, and pH cycling.

Water-soluble, endohedral gadofullerenes exhibit considerably higher relaxivities than clinically used Gd 3+chelates and are currently explored as potential magnetic resonance imaging (MRI) contrast agents. The relaxivities of Gd@C 60 (OH) x (x ≈ 27) and Gd@C 60 [C(COOH y Na 1-y ) 2 ] 10 were previously found to vary with pH because of pH-dependent aggregation. By relaxometric measurements, we proved that aggregation can be suppressed by salt addition (75-100 equiv of sodium phosphate). In the aim of better understanding paramagnetic relaxation mechanisms in water-soluble gadofullerenes, we recorded variable-temperature and multiple-field 17 O and 1 H relaxation rates for Gd@C 60 (OH) x and Gd@C 60 [C(COOH y Na 1-y ) 2 ] 10 in both aggregated and disaggregated state (monomers). In the aggregated solutions, the 17 O T 1 and T 2 values are very different. This proves the confinement of water molecules in the interstices of the aggregates which is more important for the OH than for the malonate derivative. The rapid exchange of these water molecules with bulk contributes to the high relaxivity of the aggregated gadofullerenes. After disruption of the aggregates into distinct gadofullerene molecules, the temperature-dependent proton relaxivities could be described as the sum of an outer-sphere and an inner-sphere-like mechanism. The inner-sphere-like term originates from proton exchange between the bulk and protonated OH or COOH sites. The relaxivity peak observed between 10 and 300 MHz in the nuclear magnetic relaxation dispersion (NMRD) profile evidences that the malonate groups are at least partially protonated at pH 7.4. The rotational correlation times are long (∼1.2 ns) and identical for the two gadofullerenes. The larger relaxivity of Gd@C 60 (OH) x as compared to Gd@ C 60 [C(COOH x Na 1-x ) 2 ] 10 at frequencies above 20 MHz is related to the larger number of protonated sites.

The only currently available method to measure brain glycogen in vivo is 13 C NMR spectroscopy. Incorporation of 13 C-labeled glucose (Glc) is necessary to allow glycogen measurement, but might be affected by turnover changes. Our aim was to measure glycogen absolute concentration in the rat brain by eliminating label turnover as variable. The approach is based on establishing an increased, constant 13 C isotopic enrichment (IE).13 C-Glc infusion is then performed at the IE of brain glycogen. As glycogen IE cannot be assessed in vivo, we validated that it can be inferred from that of N-acetyl-aspartate IE in vivo: After [1-13 C]-Glc ingestion, glycogen IE was 2.2 ± 0.1 fold that of N-acetyl-aspartate (n = 11, R 2 = 0.77). After subsequent Glc infusion, glycogen IE equaled brain Glc IE (n = 6, paired t-test, p = 0.37), implying isotopic steady-state achievement and complete turnover of the glycogen molecule. Glycogen concentration measured in vivo by 13 C NMR (mean ± SD: 5.8 ± 0.7 lmol/g) was in excellent agreement with that in vitro (6.4 ± 0.6 lmol/ g, n = 5). When insulin was administered, the stability of glycogen concentration was analogous to previous biochemical measurements implying that glycogen turnover is activated by insulin. We conclude that the entire glycogen molecule is turned over and that insulin activates glycogen turnover.

The relaxivity of commercially available gadolinium (Gd)-based contrast agents was studied for X-nuclei resonances with long intrinsic relaxation times ranging from 6 s to several hundred seconds. Omniscan in pure 13 C formic acid had a relaxivity of 2.9 mM À1 s À1, whereas its relaxivity on glutamate C1 and C5 in aqueous solution was~0.5 mM À1 s À1 . Both relaxivities allow the preparation of solutions with a predetermined short T 1 and suggest that in vitro substantial sensitivity gains in their measurement can be achieved. 6 Li has a long intrinsic relaxation time, on the order of several minutes, which was strongly affected by the contrast agents. Relaxivity ranged from~0.1 mM À1 s À1 for Omniscan to 0.3 for Magnevist, whereas the relaxivity of Gd-DOTP was at 11 mM À1 s À1 , which is two orders of magnitude higher. Overall, these experiments suggest that the presence of 0.1-to 10-AM contrast agents should be detectable, provided sufficient sensitivity is available, such as that afforded by hyperpolarization, recently introduced to in vivo imaging. D