Comparison of the Macromolecular MR Contrast Agents with

Medicine and Diagnostic Imaging, and Department of Radiology, Kyoto ... Department of Radiology, Otsu Municipal Hospital, Otsu 520-0804, Japan, and ...
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Bioconjugate Chem. 2001, 12, 100−107

Comparison of the Macromolecular MR Contrast Agents with Ethylenediamine-Core versus Ammonia-Core Generation-6 Polyamidoamine Dendrimer Hisataka Kobayashi,*,† Noriko Sato,‡ Satomi Kawamoto,§ Tsuneo Saga,‡ Akira Hiraga,| Tabassum Laz Haque,‡ Takayoshi Ishimori,‡ Junji Konishi,‡ Kaori Togashi,† and Martin W. Brechbiel⊥ Hitachi Medical Co. chaired Department of Diagnostic and Interventional Imagiology, Department of Nuclear Medicine and Diagnostic Imaging, and Department of Radiology, Kyoto University, Kyoto 606-8507, Japan, Department of Radiology, Otsu Municipal Hospital, Otsu 520-0804, Japan, and Chemistry Section, Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892. Received June 30, 2000; Revised Manuscript Received October 9, 2000

Two novel macromolecular MRI contrast agents based upon generation-6 polyamidoamine dendrimers (G6) of presumed similar molecular size, but of different molecular weight, were compared in terms of their blood retention, tissue distribution, and renal excretion. Two G6s with either ammonia core (G6A) or with ethylenediamine core (G6E), which possessed 192 and 256 exterior primary amino groups, respectively, were used. These dendrimers were reacted with 2-(p-isothiocyanatobenzyl)-6-methyldiethylenetriaminepentaacetic acid (1B4M). The G6-1B4M conjugates were reacted with 153Gd for studying biodistribution and blood clearance or Gd(III) for the MRI study. 3D-micro-MR angiography of the mice were taken with injection of 0.033 mmol of Gd/kg of G6A-(1B4M-Gd)192 or G6E-(1B4MGd)256 using a 1.5-T superconductive MRI unit. Although numerous fine vessels of ∼100 µm diameter were visualized on subtracted 3D-MR-angiography with both G6A-(1B4M-Gd)192 and G6E-(1B4MGd)256, 153Gd-labeled saturated G6E-(1B4M)256 remained in the blood significantly more than 153Gdlabeled saturated G6A-(1B4M)192 at later than 15 min postinjection (p < 0.01). In addition, G6E(1B4M-Gd)256 visualized these finer vessels longer than G6A-(1B4M-Gd)192. The G6A-(1B4M-Gd)192 showed higher signal intensity in the kidney on the dynamic MR images and brighter kidney images than G6E-(1B4M-Gd)256. In conclusion, the G6A-(1B4M-Gd)192 was observed to go through glomerular filtration more efficiently than G6E-(1B4M-Gd)256 resulting faster clearance from the blood and higher renal accumulation, even though both of G6-1B4M conjugates have almost similar molecular size and same chemical structure. In terms of the ability of intravascular contrast agents, G6E-(1B4MGd)256 was better due to more Gd(III) atoms per molecule and longer retention in the circulation than G6A-(1B4M-Gd)192.

INTRODUCTION

The starburst polyamidoamine (PAMAM)1 dendrimers are a new class of highly branched spherical polymers that are highly soluble in aqueous solution and have a unique surface of primary amino groups (1, 2). Compared with many other types of dendritic macromolecules that recently have been synthesized, PAMAM dendrimers are the only class of macromolecules that are unidispersed and show high positive charge densities restricted to the surface of the molecule (3). The defined structure and large number of available surface amino groups of PAMAM dendrimers have led to their use as substrates for the attachment of many chelating agents to single * To whom correspondence should be addressed. Phone: 8175-751-4955. Fax: 81-75-751-4954. E-mail: [email protected]. † Hitachi Medical Co. ‡ Department of Nuclear Medicine and Diagnostic Imaging. § Department of Radiology, Otsu Municipal Hospital. | Department of Radiology, Kyoto University. ⊥ Chemistry Section. 1 Abbreviations: PAMAM, polyamidoamine dendrimer; G6, generation-6 PAMAM dendrimer; 1B4M, 2-(p-isothiocyanatobenzyl)-6-methyl-diethylenetriamine-pentaacetic acid; DOTA, 1,4,7,10-tetraazacyclododecane tetraacetic acid; MIP, maximum intensity protection.

antibody molecule (2, 4-6) and in the preparation of novel MRI contrast agents (7, 8). Two types of macromolecular contrast agents for MRI have been reported. One was albumin conjugated with diethylenetriaminepentaacetic acid (DTPA) [albumin(DTPA-Gd)30-34], originally described by Ogan et al. (9). The other was fundamentally a polyamine conjugated with multiple chelates [DTPA derivatives or 1,4,7,10tetraazacyclododecanetetraacetic acid (DOTA) derivatives]. Although polylysine has been used for the core of contrast agents among the various polyamines (10, 11), PAMAM dendrimers have recently been preferred as an alternative to polylysine (10-12). Wiener et al. and we reported the preferable use of generation-6 PAMAM dendrimers (G6) with ammonia core (G6A) conjugated with DTPA derivative as an intravascular macromolecular MR contrast agent (7, 13). G6 with an ethylenediamine core (G6E) have almost similar molecular size and possesses more amines than G6A (256 for G6E compared with 192 for G6A) (Figure 1). Therefore, G6E fully conjugated with DTPA can potentially complex and sequester 1.33 times as many metal atoms as G6A. In this study, the in vivo pharmacokinetics of 153Gdlabeled G6E-(1B4M)x were evaluated in the mice and compared with those of 153Gd-labeled G6A-(1B4M)x. In addition, the differences between G6E-(1B4M-Gd)256

10.1021/bc000075s CCC: $20.00 © 2001 American Chemical Society Published on Web 12/28/2000

Comparison of Pharmakokinetics of Different Generation-6 Dendrimers

Figure 1. Scheme of the two core types of PAMAM dendrimers.

and G6A-(1B4M-Gd)192 as MR contrast agents were evaluated. EXPERIMENTAL PROCEDURES

PAMAM Dendrimers. The generation-6 PAMAM dendrimer with an ammonia core (G6A) (Polysciences, Warrington, PA) that possesses 192 amino groups and has a molecular mass of 43 451 Da and the generation-6 PAMAM dendrimer (G6E) (Aldrich Chemical Co., Milwaukee, WI) with an ethylenediamine core that possesses 256 reactive amino groups and has a molecular mass of 57 991 Da were used. Conjugation of Chelates to G6 Dendrimers. The dendrimers were concentrated to ∼5 mg/mL and diafilterated against 0.1 M phosphate buffer at pH 9. The G6A and G6E dendrimers were reacted with a 192 or a 256fold molar excess of 2-(p-isothiocyanatobenzyl)-6-methyldiethylenetriaminepentaacetic acid (1B4M) at 40 °C, respectively, and maintained at pH 9 with 1 M NaOH for 24 h. Another additional equal amount of the 1B4M was added to each after 24 h as a solid. The resulting preparations were purified by diafiltration using a Centricon 30 (Amicon Co., Beverly, MA) for either G6A or G6E. This resulted in 99.2 ( 1.1 and 99.8 ( 1.5% of the amine groups on the G6A and G6E dendrimers reacting with the 1B4M for four different preparations of each core, respectively, as determined by 111In (Mediphysics, Takarazuka, Japan) labeling of the reacted samples as previously described (14). In brief, ∼500 000 cpm of 111 In acetate was added to ∼100 µg/10 µL of the conjugate samples before diafiltration and incubated in 0.2 M acetate buffer for 15 min at room temperature. Then, the bound fractions to the conjugates and free 1B4M were separated using a PD-10 column (Pharmacia) and counted. After which, the numbers of 1B4M chelates bound to both G6A and G6E were calculated, respectively, from the count of the radioactivity in the bound fraction to the conjugates. To evaluate the effect of fully saturating the amino groups on the surface of G6s with 1B4M chelates on their biodistribution properties, G6A and G6E were each reacted with a 4-fold molar excess of 1B4M under the same conditions described above. Finally, after purification as related above we isolated the G6A-1B4M2 and G6E-1B4M2 conjugates. Assaying again by 111In confirmed saturation of the surface of the dendrimers with the 1B4M chelatng agent. Radiolabeling of G6-1B4Ms. Carrier-free 153GdCl3 was purchased from NEN DuPont (Boston, MA) and ∼750 µg (1 µmol) of each G6-1B4M conjugate was reacted with 20 µCi of 153Gd citrate in 0.3 M citrate buffer at pH 5 for 30 min at room temperature. To remove any nonincorporated free radiometal, 10 µL of 0.5 M EDTA

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(Nakarai, Tokyo, Japan) was added and the product was purified by PD-10 column (Pharmacia, Uppsala, Sweden), which was eluted with PBS, (pH 7.4). The resulting purified materials were referred to as “unsaturated” because on the average they had one radiometal (153Gd) per total molecule. To evaluate the effect on biodistribution by saturation of the 1B4M chelating agents with metals, the prepared G6-1B4M conjugateswere each saturated with nonradioactive gadolinium. The preparation method was the same as described above, except that a 3-fold molar excess of nonradioactive Gd(III) citrate (Nakarai, Tokyo, Japan) (3 µmol) to conjugated 1B4M was added to the reaction mixture and incubated for 1 h before addition of EDTA and subsequent column purification. The radiolabeling yields were 95-97% and from ∼40%, respectively, for the unsaturated and saturated preparations. Preparation of Contrast Agent for MRI with nonrAdioactive Gd(III). Approximately ∼3 mg of each G6-1B4M conjugate (containing 4 µmol of 1B4M) were mixed with 6 µmol of nonradioactive Gd(III) citrate in 0.3 M citrate buffer for 2 h at 40 °C. The excess Gd in each preparation was removed by diafiltration using the Centricon 30 (Amicon Co.) for G6A and G6E while simultaneously changing the buffer to 0.05 M PBS. The purified samples were diluted to 0.5 mL with 0.05 M PBS and 100 µL was used as the MRI contrast agent per mouse. A replacement assay for three different preparations of each conjugate using 153Gd determined that 81.9 ( 1.9 and 82.5 ( 1.8% of the 1B4M on the G6D-1B4M conjugates were indeed chelating Gd(III) atoms, respectively. In brief, ∼100 000 cpm of 153Gd was added with 0.1 µmol of nonradioactive Gd(III) to 5 µL of the injected samples and incubated in 0.5 M citrate buffer for 2 h at 40 °C, after which the bound and unbound fractions to the conjugates were separated using a PD-10 column (Pharmacia) and counted. After which, the number of Gd atoms replaced from the gadolinium citrate to 1B4M was calculated from the count of the radioactivity in the bound fraction. Then, the number of available 1B4M chelates for binding Gd atoms on the conjugates was calculated. Biodistribution of 153Gd-Labeled G6-1B4Ms. Six groups of nude mice (n ) 4 in each group) were each injected with one of the following preparations: 0.8 µCi/ 200 µL of 153Gd-labeled unsaturated G6A-(1B4M)192 or G6E-(1B4M)256; 0.5 µCi/200 µL of 153Gd-labeled saturated G6A-(1B4M)192 or G6E-(1B4M)256; 0.5 µCi/200 µL of 153Gd-labeled saturated G6A-(1B4M)2 or G6E(1B4M)2. All studies were approved by the Animal Care Committee of Kyoto University. The mice were euthanized 15 min after the injection of 153Gd-labeled preparation. Biodistribution studies were carried out, and the data were expressed as the percent injected dose per gram (% ID/g) of tissue and blood-to-normal tissue ratio. The blood was drawn (∼0.7 mL) by the cardiac puncture. In the case of bone, the bone marrow was included. Blood Clearance of 153Gd-Labeled Saturated G61B4Ms. Two groups of nude mice (n ) 4 in each group) were carefully injected with 3.5 µCi/200 µL of 153Gdlabeled saturated G6A-(1B4M)192 or G6E-(1B4M)256 from the proximal portion of the left tail vein without any subcutaneous leakage. All mice received a small cut on their distal or middle portion of the right tail vein and blood samples were collected with a 5-µL micropipet 2, 6, 15, 30, 60, 120, and 240 min postinjection of 153Gdlabeled preparations. The data were expressed as the

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Figure 2. Biodistribution of 153Gd-labeled unsaturated or saturated preparations of G6A [G6A-(1B4M-Gd)2 (9), G6A-(1B4M)192Gd (cross-hatched bar), or G6A-(1B4M-Gd)192 (0)] (a) and 153Gd-labeled unsaturated or saturated preparations of G6E [G6E-(1B4MGd)2 (9), G6E-(1B4M)256-Gd (cross-hatched bar), or G6E-(1B4M-Gd)256 (0)] (b) in normal nude mouse at 15 min after injection. The data are expressed as the mean percentages of injected dose per gram of normal tissues and standard deviation (n ) 4 or 5). The asterisks indicate significant differences (p < 0.01) compared with G6A.

Figure 3. Blood clearance of 153Gd-labeled saturated G6A(1B4M-Gd)192 (open circle) or G6E-(1B4M-Gd)256 (closed circle) in normal nude mouse 2, 6, 15, 30, 60, 120, and 240 min postinjection. The data are expressed as the mean percentages of injected dose per gram of blood and standard deviation (n) 4). The asterisks indicate significant differences (p < 0.01) compared with G6A.

percent injected dose per gram (% ID/g) of blood, and then the data of each mouse were fitted by the singleexponential curve, and the half-life of each preparation from the blood was calculated. Excretion and Body Retention of 153Gd-Labeled Saturated G6D-1B4M Conjugates. Two groups of nude mice (n ) 3 in each group) were injected with 2 µCi/200 µL of either 153Gd-labeled saturated G6A(1B4M)192 or G6E-(1B4M)256. The mice were put in a metabolic cage for 2 days and their urine and feces were serially collected 3, 10, 24, and 48 h postinjection. The mice were euthanized and the amount of 153Gd in the carcass was measured. The data were expressed as percentage of injected dose (% ID).

Figure 4. Urinary (circle) and fecal (squear) excretion of 153Gd-labeled G6A-(1B4M-Gd) 192 (open circle/squear), or G6E(1B4M-Gd)256 (closed circle/squear). The data are expressed as the mean percentages of injected dose of three mice, which put in a metabolic cage together.

3D-micro-MR Angiography of Mice. MR angiography of the mice were taken with injection of 0.033 mmol of Gd/kg of G6D-A-(1B4M-Gd)192 (175 kDa) or G6D-E(1B4M-Gd)256 (240 kDa) using a 1.5-T superconductive magnet unit (Signa, General Electric Medical System, Milwaukee, WI). Six female 8 week-old balb/c nu/nu mice of 18-21 g body weight in each group were used and each contrast agent was prepared at least four separate times for these imaging studies. All images were obtained with dual phased-array 3-in. round surface coils fixed at 3-cm intervals by an in-house constructed mouse and coil holder. The mice were anesthetized with 1.15 mg of sodium pentobarbital (Dainabot, Osaka, Japan) and placed at the center of the coils. The 3D-fast spoiled gradient echo (FSPGR; TR/TE 150/4.2; flip angle 60°; scan time 1 min, 48 s) with chemical fat-suppression

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Figure 5. Plots of the signal intensity in the left ventricle of the heart (a), the cortex (b) and medulla (c) of the kidney, the inferior vena cava (d), the liver (e), and the muscle (f) obtained from the contrast-enhanced dynamic MRI of mice with 0.033 mmol/Kg of G6A-(1B4M-Gd)192 (open circle) or G6E-(1B4M-Gd)256 (closed circle). The single and double asterisks indicate significant differences (p < 0.01 and p < 0.05, respectively).

technique were used for all mice from 0 to 14 min after injection of the contrast agents for all mice. The coronal images were reconstructed with 2-mm section thickness without gaps. The FOV was 8 × 6 cm and the size of matrix was 256 × 192. The regions of interest were set to be the ventricles, inferior vena cava, the cortex and medulla of the kidney, and other organs and analyzed the time-intensity curve. In addition, the slice data were processed subtracting from postcontrast images to precontrast images and constructing into 3D images with the maximum intensity protection (MIP) method using

a work station (Advantage Windows, General Electric Medical System). Statistical Analysis. Statistical analysis was performed using either student-t test or the one-way analysis of variance (ANOVA), with pair wise comparison using the Bonferroni method (Sigmastat, Jandel Scientific, San Rafael, CA). RESULTS

Biodistribution of 153Gd-Labeled G6-1B4Ms. Saturated G6E vs G6A. The 153Gd-labeled saturated G6E-

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Figure 6. The values of the enhancement effects ratio in the cortex (a) and the medulla (b) of kidney, the liver (c), and the muscle (d) to the blood in the left ventricle of the heart were plotted. (enhancement effects ratio) ) {[(the postcontrast signal intensity of the organs) - (the precontrast signal intensity of the organs)]/[(the postcontrast signal intensity of the left ventricle) - (the precontrast signal intensity of the left ventricle)]} The single and double asterisks indicate significant differences (p < 0.01 and p < 0.05, respectively).

(1B4M)256 (32.4 ( 0.6% ID/g) remained in the blood significantly more than the 153Gd-labeled saturated G6A(1B4M)192 (24.2 ( 1.1% ID/g) (p < 0.01) and accumulated significantly less in the liver (17.1 ( 1.2 vs 28.9 ( 1.2% ID/g) and in the kidney (13.6 ( 0.5 vs 17.8 ( 0.4% ID/g) than all other preparations (p < 0.01) (Figure 2). Saturated G6E/G6A vs Unsaturated G6E/G6A. Both 153 Gd-labeled unsaturated G6-(1B4M)x preparations accumulated significantly more in the liver (51.6 ( 2.4 and 57.2 ( 1.2% ID/g for G6E and G6A) and less in the blood (0.4 ( 0.1 and 0.3 ( 0.1% ID/g for G6E and G6A) than the saturated preparations (p < 0.01) (Figure 2). G6-(1B4M)2 Preparation. Both 153Gd-labeled saturated G6-(1B4M)2 preparations which have much smaller molecular masses (60 and 45 kDa for G6E and G6A), accumulated significantly less in the liver (24.4 ( 0.5 and 25.7 ( 0.9% ID/g for G6E and G6A) and more in the kidney (59.8 ( 0.5 and 78.7 ( 2.7% ID/g for G6E and G6A) than both the 153Gd-labeled unsaturated and saturated G6E(1B4M)256 and 153Gd-labeled G6A-(1B4M)192 (p < 0.01) (Figure 2). In this case, 153Gd-labeled saturated G6E(1B4M)2 accumulated significantly less in the kidney than 153Gd-labeled saturated G6A-(1B4M)2 (p < 0.01). Blood Clearance of 153Gd-Labeled Saturated G61B4Ms. The blood retention of 153Gd-labeled G6E(1B4M)256 was significantly greater than that of 153Gdlabeled G6A-(1B4M)192 at all time points later than 15 min postinjection (Figure 3; p < 0.01). The blood halflife of the 153Gd-labeled saturated PAMAM-1B4Ms was

132 ( 29 and 51 ( 17 min for G6E-(1B4M-Gd)256 and G6A-(1B4M-Gd)192, respectively. Excretion and Body Retention of 153Gd-Labeled Saturated G6D-1B4M Conjugates. The cumulative urinary excretion of 153Gd-labeled G6E-(1B4M)256 (6.0% ID) was less than that of 153Gd-labeled G6A-(1B4M)192 (8.5% ID) within 48 h postinjection (Figure 4). The cumulative fecal excretion of 153Gd-labeled G6E-(1B4M)256 (3.4% ID) was more than that of 153Gd-labeled G6A(1B4M)192 (1.9% ID) within 48 h postinjection (Figure 4). The whole body retention of 153Gd-labeled G6E(1B4M)256 and 153Gd-labeled G6A-(1B4M)192 was 80.9 ( 2.8 and 79.7 ( 3.2% ID 48 h postinjection, respectively (p ) 0.6). 3D-micro-MR Angiography of Mice. G6E-(1B4MGd)256 showed slightly higher signal intensity in the left ventricle of the heart and the inferior vena cava at some of the later time points and significantly lower signal intensity than G6A-(1B4M-Gd)192 in the both cortex and medulla of the kidney at all time points that were examined (p < 0.01; Figure 5). No significant difference in the signal intensity was observed in the liver and the muscle. Therefore, left ventricle/kidney signal intensity ratio of G6E-(1B4M-Gd)256 was significantly lower than that of G6A-(1B4M-Gd)192 (p < 0.01; Figure 6). The subtraction MR-angiography with G6A-(1B4MGd)192 showed apparently brighter kidneys on both early and delayed images (Figure 7).

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Figure 7. A pair of the whole body 3D-micro-subtraction-MR-angiography of a mouse, which were constructed with the MIP method with immediately after (left) and 8 min after (right) injection of 0.033 mmol of Gd/kg of G6A-(1B4M-Gd)192 (a) or G6E-(1B4MGd)256 (b) are shown. The brighter kidney was shown on panel a than on panel b. The scale shown at the bottom indicates 1 cm. DISCUSSION

The difference in molecular diameter between G6E(1B4M-Gd)256 and G6A-(1B4M-Gd)192 was theoretically expected to be very small because the only difference between those molecules were the core portions (ammonia versus ethylenediamine). Both of these molecules were expected then to have arms of the same length and thus similar spherical shape. When the 1B4M was conjugated with all amino-groups on the surface of the

molecule, the chemical properties of both molecules were then expected to remain similar. However, not only G6E(1B4M-Gd)256 versus G6A-(1B4M-Gd)192, but also G6E(1B4M-Gd)2 versus G6A-(1B4M-Gd)2, showed different pharmacokinetics in vivo, especially in the kidney (glomerular filtration). The G6A was predicted to have sparser arms than G6E simply on a numerical basis. Therefore, we speculated that G6A-(1B4M-Gd)192 would be more flexible for changing shape than G6E-(1B4M-Gd)256,

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resulting in increasing the possibility to pass through glomerular filtration. In the previous and current studies, supporting data was obtained to explain that not only the molecular weight, but also the size and shape of the molecule is an important contributing criteria for glomerular filtration (13). In brief, a hypothesis that the PAMAM-(1B4MGd)x might be filtered through the glomerulus more readily than a nonspherical protein with a comparable molecular weight was formed. Previous results indicated that the G5A-(1B4M-Gd)96 (88 kDa) was partially filtered through the glomerulus, even though G5A-(1B4MGd)96 had a greater molecular mass than albumin, thus supporting this hypothesis. In addition, although G4E(1B4M-Gd)64 (57 kDa) had greater molecular mass and smaller molecular size than G6A-(1B4M-Gd)2 (45 kDa), the G4E-(1B4M-Gd)64 was significantly filtered more from the glomerulus than G6A-(1B4M-Gd)2. From these results, not only molecular mass, but also shape, size or charge of the molecule affects the glomerular filtration of these molecules. A second aspect of this study was that differences in the surface charge density of these molecules might also be an important criteria influencing both clearance rate and route. G6E should have higher surface density charge than G6A simply due to the greater number of 1B4Ms on the exterior of a similar size spherical surface. We have previously reported that the charge of two molecules of similar molecular size, ranging from 40 to 70 kDa, can strongly affect their glomerular filtration (15). However, in this case, this explanation would not be appropriate. The unsaturated G6E-(1B4M-Gd)256, which possesses a greater negative charge and G6E(1B4M-Gd)2, which has a greater positive charge, both exhibited lower renal accumulation than their respective G6A counterparts. The differences in glomerular filtration influenced the blood clearance rate for both molecules as well, which was reflected in their ability to function as macromolecular intravascular MR contrast agent. Macromolecular contrast agents for magnetic resonance imaging (MRI) have been reported to be excellent methods to evaluate microvasculature and histological capillary density in tumor tissues (16, 17). Although postcontrast signal changes in the tumors have shown better correlation with histological capillary mass, only in our previous report was the intratumoral microvasculature directly evaluated with MRI using macromolecular MR contrast agents (Kobayashi et al. in submission to Magn. Reson. Med.) In terms of prolongation of contrast-enhancement and large number of Gd(III) atoms containing in a molecule, G6E-(1B4M-Gd)256 is superior to G6A-(1B4M-Gd)192. Additionally, when using the G6A-(1B4M-Gd)192 to visualize microvasculature clearly, the dose of Gd(III) atoms could be decreased to 33% of the current clinical dose for Gd-DTPA (13). Thus, use of G6E-(1B4M-Gd)256 would possibly decrease the dose of Gd(III) minimizing toxicity. In conclusion, G6E-(1B4M-Gd)256 demonstrated longer blood retention and lower renal accumulation than the G6A-(1B4M-Gd)192, although both of these molecules were expected to theoretically have approximately the same molecular diameter. G6E-(1B4M-Gd)256 may be a potential candidate as an intravascular MR contrast agent with a maximum number of Gd(III) atoms and minimal size so as not to be excreted through the kidney and perform as a superior contrast agent for vascular imaging.

Kobayashi et al. ACKNOWLEDGMENT

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