Polyaspartamide Gadolinium Complexes Containing Sulfadiazine

Jul 6, 2005 - Magnetic Resonance Imaging (MRI) Contrast Agents for Tumor Diagnosis. Weiren Cheng , Yuan Ping , Yong Zhang , Kai-Hsiang Chuang , Ye Liu...
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Bioconjugate Chem. 2005, 16, 967−971

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Polyaspartamide Gadolinium Complexes Containing Sulfadiazine Groups as Potential Macromolecular MRI Contrast Agents Guo-Ping Yan,*,† Mai-Li Liu,‡ and Li-Yun Li‡ School of Materials Science and Engineering, Wuhan Institute of Technology, Wuhan 430073, P. R. China, and State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Institute of Physics and Mathematics, The Chinese Academy of Sciences, Wuhan 430071, P. R. China. Received February 1, 2005; Revised Manuscript Received May 16, 2005

Both diethylenetriaminepentaacetic acid (DTPA) and sulfadiazine (SD) were incorporated into polyaspartamides with different side chains, including poly-R,β-[N-(2-hydroxyethyl)-L-aspartamide] (PHEA), poly-R,β-[N- (3-hydroxypropyl)-L-aspartamide] (PHPA), poly-R,β-[N-(2-aminoethy1)-L-aspartamide] (PAEA), poly-R,β-[N-(4-aminobuty1)-L-aspartamide] (PABA), and poly-R,β-[N-(6-aminohexyl)L-aspartamide] (PAHA). The polyaspartamide ligands containing DTPA and SD groups were further reacted with gadolinium chloride to give the corresponding macromolecular gadolinium complexes. Experimental data of 1H NMR, IR, UV, and elemental analysis exhibited the formation of the polyaspartamide ligands and gadolinium complexes. Relaxivity studies indicated that the macromolecular chelates possess higher relaxivities than that of the clinically used Gd-DTPA. MR imaging showed that the macromolecular chelate PAEA-Gd-DTPA-SD greatly enhanced the contrast of MR images of hepatoma in the lower limb of mice and provided prolonged intravascular duration. Thus the polyaspartamide gadolinium complex containing SD groups is expected to be used as the potential macromolecular MRI contrast agents for hepatoma in mice.

INTRODUCTION

Magnetic resonance imaging (MRI) is a noninvasive clinical imaging modality, which relies on the detection of NMR signals emitted by hydrogen protons in the body placed in a magnetic field. MRI contrast agent is a unique class of pharmaceutical that enhances the image contrast between normal and diseased tissue and indicates the status of organ function or blood flow after administration by increasing the relaxation rates of water protons in tissue in which the agent accumulates (1-3). The clinically used MRI contrast agents, for example, gadolinium diethylenetriaminepentaacetic acid (GdDTPA), are small ionic molecules that can diffuse freely through the extracellular space and are excreted rapidly by the kidney. Their biodistribution is nonspecific although Gd-DTPA works well in the brain and spinal cord (4, 5). The binding of small gadolinium complex to a macromolecule has been shown to provide a mechanism of proton relaxation enhancement through longer rotational correlation times. The macromolecular MRI contrast agent thus obtained may show prolonged intravascular retention due to the bulky molecular volume, resulting in the clinical use as a blood pool contrast agent. In addition, when a tumor-targeting group is attached to this macromolecular metal chelate, it can be endowed with tumor-specific property (3, 6). Polyaspartamide is a water-soluble, biologically welltolerated synthetic polymer with a protein-like structure that was used as a plasma extender and a drug carrier because it is nontoxic, nonantigenic, and degradable in living systems and modified readily by the reaction with * To whom correspondence should be addressed. E-mail: [email protected]. † Wuhan Institute of Technology. ‡ The Chinese Academy of Sciences.

the side chain (7, 8). As reported, antiviral drugs and antiinflammatory agents were covalently linked to polyR,β-[N-(2-hydroxyethyl)-D,L-aspartamide] (PHEA) forming drug-polymer conjugates capable of increasing drug stability and bioavailility. Therefore polyaspartamide can be used as polymer carriers for MRI contrast agents (912). Sulfadiazine derivatives were reported to be concentrated into Walker carcinoma or Yoshida sarcoma by a factor of about 2-3 compared with the uptake in the liver (13-14). Previously we have used sulfadiazine (SD) as a tumor-selective group and evaluated Gd-DTPA derivatives containing SD groups as the potential tumor-specific contrast agents. The biodistribution and MRI studies indicated that the incorporation of SD into Gd-DTPA increased their uptake by hepatoma and Ehrlich ascites carcinoma in mice, and the gadolinium complexes exhibited tumor-selective properties (15). In this work, the polyaspartamide ligands were synthesized by the incorporation of both DTPA and SD into polyaspartamides with different side chains. The ligands were further reacted with gadolinium chloride to give the corresponding polyaspartamide gadolinium complexes (Scheme 1). The polyaspartamide ligands and gadolinium complexes were characterized by 1H NMR, IR, UV, and elemental analysis. Relaxivity and magnetic resonance imaging of hepatoma (H22) in mice were evaluated. EXPERIMENTAL PROCEDURES

Instrumentation and Materials. The compounds prepared were characterized using a Spectrum One infrared spectrophotometer, a Lambda Bio40 UV/Vis spectrophotometer, a Varian Mercury-VX300 NMR spectrometer, and a Carlo Erba 1106 analyzer. The molecular weight and polydispersity of polyaspartamide were measured by GPC [software version millemmium 32, Waters

10.1021/bc050026l CCC: $30.25 © 2005 American Chemical Society Published on Web 07/06/2005

968 Bioconjugate Chem., Vol. 16, No. 4, 2005 Scheme 1. Synthetic Route to Polyaspartamide MRI Contrast Agents

2960D separation module, Ultrahydrogel 2000 column, poly(Ethylene Glycol) standard, 0.1 M KH2PO4 solvent, 0.5 mL‚min-1 flow, 35 °C column temperature and 30 °C detector temperature]. The concentration of the paramagnetic species [Gd3+] was measured by an ICP Atomscan-2000 spectrometer. The solvent longitudinal relaxation time (T1) for gadolinium complexes in distilled water was determined by a Varian Mercury-VX300 NMR spectrometer. MR image was performed on a 4.7 T BIOSPEC 47/30 MR Scanner (Bruker Medical, Ettlingen, Germany). KunMing mice bearing with hepatoma (H22) in the right lower limb (weight: 30 ( 2 g) were provided by the Department of Pharmacy (School of Medicine, Huazhong University of Science and Technology, China) and were cultured according to the method described in the literature (16). Hepatoma (H22) cells were provided by the China Center for Type Culture Collection of Wuhan University, China. All the chemicals and solvents were of analytical grade. The solution of phosgene in toluene (2.0 M) (17), DTPA active ester including DTPA mono(carbonyloxyisobutyl ester) (i-BuOCO-DTPA) and DTPA mono(hydroxysuccinimidyl ester) (SuO-DTPA) (18), poly-R,β-[N-(2-hydroxyethyl)-L-aspartamide] (PHEA), poly-R,β-[N-(2-aminoethyl)L-aspartamide] (PAEA), poly-R,β-[N-(3-hydroxypropyl)L-aspartamide] (PHPA), poly-R,β-[N-(4-aminobutyl)-Laspartamide] (PABA), and poly-R,β-[N-(6-aminohexyl)L-aspartamide] (PAHA) (19) were prepared according to the literature. Synthesis of Polyaspartamide Ligands. Synthesis of PHEA-DTPA-SD and PHPA-DTPA-SD. Sulfadiazine (SD, 1.0 g, 4 mmol) was dissolved in 20 mL of N,Ndimethylformamide (DMF) and 5 mL of pyridine. The mixture was added slowly to a solution of phosgene in toluene (10 mL) with rapid stirring at -10 °C to -5 °C. The reaction was stirred for 2 h at this temperature and a further 2 h at 0 °C under an argon atmosphere. After filtration, a brown solution of SDCOCl in DMF was obtained. The solution of SDCOCl (4 mmol) in DMF obtained above was added slowly to a solution of PHEA (1.58 g, 10 mmol) in 40 mL of DMF and 8 mL of triethylamine with rapid stirring at 0 °C. The reaction was stirred for 4 h at 0 °C and a further 24 h at room temperature. After filtration, the filtrate was collected and cooled to -5 °C. The solution of DTPA mono(carbonyloxyisobutyl ester) (i-BuOCO-DTPA, 3.82 g, 8 mmol) in DMF (20 mL) was

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added dropwise to the filtrate at -5 °C. The reaction was continued stirring for 4 h at -5 °C and a further 48 h at room temperature. The resultant mixture was filtered and precipitated with ethanol and ethyl ether. The precipitate was reprecipitated from DMF using ethanol and ethyl ether, filtered, and dried under vacuum to yield a white polyaspartamide ligands containing DTPA and SD groups PHEA-DTPA-SD (M1) (2.0 g, 30.1%). 1H NMR (D2O, δ, ppm): 8.1-7.7 (C6H4, C4N2H2), 4.5, 4.2, 4.0 (CH, CH2), 3.7(CH2CO), 3.4-3.3(NCH2CH2N), 3.2, 3.0, 2.8, 2.6-2.4 (NHCH2, CH2); IR(KBr, cm-1): 3426.6 (OH), 2975.7-2492.1 (C-H), 1711(COO), 1634.7(CONH), 1397.2, 1172.5(SdO), 1154.6(C-N), 1094.2 (C-O), 907.2, 840.3, 803.8(C6H4, C4N2H2); UV(H2O, nm): 272.9. The polyaspartamide ligands containing differing amounts of DTPA linked to polymeric repeat units were synthesized by the same method to give the following results PHEA-DTPA-SD (M2, 28%) and PHPA-DTPA-SD (32%). PHEA-DTPA-SD (M2): 1H NMR (D2O, δ, ppm): 8.17.7 (C6H4, C4N2H2), 4.5, 4.2, 4.0 (CH, CH2), 3.7(CH2CO), 3.4-3.3(NCH2CH2N), 3.2, 3.0, 2.8, 2.6-2.4 (NHCH2, CH2); IR(KBr, cm-1): 3436.1(OH), 2975.4-2492(C-H), 1710(COO), 1637.4(CONH), 1474.2, 1433.9(C6H4, C4N2H2), 1397.9, 1171.5(SdO), 1229.1(C-N), 1036.2 (C-O), 910.7, 849.1, 804.4(C6H4, C4N2H2); UV(H2O, nm): 272.9. PHPA-DTPA-SD: 1H NMR (D2O, δ, ppm): 8.2-7.7(C6H4, C4N2H2), 4.5, 4.2, 4.0 (CH, CH2), 3.7(CH2CO), 3.43.2(NCH2CH2N), 3.0, 2.8, 2.6-2.4 (NHCH2, CH2); IR(KBr, cm-1): 3434.4(OH), 2975.3-2491.9(C-H), 1713(COO), 1636.0(CONH), 1474.5, 1434.1(C6H4, C4N2H2), 1397.9, 1171.3(SdO), 1229.4(C-N), 1036.2 (C-O), 909.8, 849.1, 804.4(C6H4, C4N2H2); UV(H2O, nm): 268. Synthesis of PAEA-DTPA-SD, PABA-DTPA-SD and PAHA-DTPA-SD. The solution of SDCOCl (4 mmol) in DMF obtained above was added slowly to a solution of PAEA (1.57 g, 10 mmol) in 100 mL of distilled water and 8 mL of triethylamine with rapid stirring at -5 °C. The reaction was stirred for 4 h at -5 °C and a further 24 h at room temperature. After filtration, the filtrate was collected and cooled to -5 °C. The solution of DTPA mono(hydroxysuccinimidyl) ester (SuO-DTPA, 3.92 g, 8 mmol) in DMF (20 mL) was added dropwise to the filtrate at -5 °C. The reaction was continued stirring for 4 h at -5 °C and a further 48 h at room temperature. The resultant mixture was filtered and precipitated with ethanol and ethyl ether. The precipitate was reprecipitated from distilled water using ethanol and ethyl ether, filtered, and dried under vacuum to yield a white polyaspartamide ligands containing DTPA and SD groups PAEA-DTPA-SD (M1) (2.1 g, 31.2%). 1H NMR (D2O, δ, ppm): 8.0-7.6 (C6H4, C4N2H2), 4.5, 4.2, 4.0 (CH, CH2), 3.7(CH2CO), 3.4-3.1(NCH2CH2N), 3.0-2.8, 2.8-2.4, 2.42.2 (NHCH2CH2NH); IR(KBr, cm-1): 3433.4(NH2), 3071.22932.3(C-H), 1711(COO), 1644.4, 1545.4(CONH), 1435.3(C6H4, C4N2H2), 1398.7, 1171.7(SdO), 1321.5(C-N), 1090.3(C-O), 912.4, 849.1, 800.4(C6H4, C4N2H2); UV(H2O, nm): 301.8. The polyaspartamide ligands containing differing amounts of DTPA linked to polymeric repeat units were synthesized by the same method to give the following results PAEA-DTPA-SD (M2, 29.2%), PABA-DTPA-SD (33%) and PAHA-DTPA-SD (33.5%). PAEA-DTPA-SD (M2): 1H NMR (D2O, δ, ppm): 8.07.6 (C6H4, C4N2H2), 4.5, 4.2, 4.0 (CH, CH2), 3.8(CH2CO), 3.6-3.2(NCH2CH2N), 3.2-3.0, 2.8, 2.6-2.4 (NHCH2CH2NH); IR(KBr, cm-1): 3398.2(NH2), 3057.3-2927.6(C-H), 1709(COO), 1648.1, 1535.3(CONH), 1432(C6H4, C4N2H2),

Polyaspartamide Gadolinium Complexes

1394.8, 1174.0(SdO), 1236.9(C-N), 1088.1(C-O), 909.1, 848.1, 800.4(C6H4, C4N2H2); UV(H2O, nm): 301.8. PABA-DTPA-SD: 1H NMR (D2O, δ, ppm): 8.1-7.6 (C6H4, C4N2H2), 4.5, 4.1, 4.0 (CH, CH2), 3.7(CH2CO), 3.43.3(NCH2CH2N), 3.0, 2.8, 2.6-2.4 (NHCH2, CH2); IR(KBr, cm-1): 3445.2(NH2), 2975.6-2491.8(C-H), 1708.6(COO), 1634.8, 1535.3(CONH), 1473.9, 1434.0(C6H4, C4N2H2), 1397.9, 1171.1(SdO), 1226.8(C-N), 1036.1(C-O), 909.1, 849.6, 805.0(C6H4, C4N2H2); UV(H2O, nm): 268.5. PAHA-DTPA-SD: 1H NMR (D2O, δ, ppm): 7.9-7.7 (C6H4, C4N2H2), 4.5, 4.1, 4.0 (CH, CH2), 3.8(CH2CO), 3.53.3(NCH2CH2N), 3.0, 2.8, 2.6-2.4, 2.2 (NHCH2, CH2); IR(KBr, cm-1): 3436.2(NH2), 3063.9-2934.2(C-H), 1714(COO), 1641.7, 1541.5(CONH), 1435.3(C6H4, C4N2H2), 1395.5, 1177(SdO), 1229.1(C-N), 1047.9(C-O), 907.8, 850, 804(C6H4, C4N2H2); UV(H2O, nm): 291. Preparation of Polyaspartamide Gadolinium Chelates. The polyaspartamide gadolinium complexes with differing amounts of Gd-DTPA linked to polymeric repeat units and differing polymers were synthesized according to the following standard procedure. PHEA-DTPA-SD (M1, 1.08 g, 5 mmol) was dissolved in 20 mL of distilled water and gadolinium chloride (GdCl3, 1.98 g, 7.5 mmol) was added. The mixture was stirred for 1 h, adjusted with 2 M NaOH solution to pH 5, and continued to stir for 12 h at room temperature. After dialysis, the dialyzed solution was evaporated and the solid residue was dried under vacuum to yield polyaspartamide gadolinium complex PHEA-Gd-DTPA-SD (M1, 1.18 g, 73.5%). PHEAGd-DTPA-SD (M2, 65%), PHPA-Gd-DTPA-SD (74%), PAEA-Gd-DTPA-SD (M1, 77%), PAEA-Gd- DTPA-SD (M2, 71%), PABA-Gd-DTPA-SD (72%) and PAHA-GdDTPA-SD (68%) were similarly synthesized. PHEA-Gd-DTPA-SD (M1): IR(KBr, cm-1): 3445.2(OH), 2932.2(C-H), 1659.0, 1642.0 (COO, CONH), 1542.1(C6H4, C4N2H2), 1411, 1155(SdO), 1261.8(C-N), 1059.8(C-O), 930.8, 868.2, 801.0(C6H4, C4N2H2). Elem. Anal.(%) found (calcd): C 42.40(42.70), N 15.59(15.70), H 5.73(5.69). The average mole ratio of attached Gd-DTPA and SD to all polymeric repeat units (mol %): Gd-DTPA 7.93 (as determined by an ICP Atomscan-2000 spectrometer), SD 0.57 (as determined by 1H NMR); UV(H2O, nm): 272.9. PHEA-Gd-DTPA-SD (M2): IR(KBr, cm-1): 3431.3(OH), 2927.7(C-H), 1646.9, 1658.3, 1555.6(COO, CONH), 1542.1, 1435.5(C6H4, C4N2H2), 1409.4, 1091.8(SdO), 1265.6(CN), 1057.2(C-O), 931.8, 867, 801(C6H4, C4N2H2). Elem. Anal.(%) found (calcd): C 40.50(40.96), N 14.33(14.49), H 5.32(5.26). The average mole ratio of attached GdDTPA and SD to all polymeric repeat units (mol %): GdDTPA 15.75 (as determined by an ICP Atomscan-2000 spectrometer), SD 0.23 (as determined by 1H NMR); UV(H2O, nm): 272.9. PHPA-Gd-DTPA-SD: IR(KBr, cm-1): 3422.3(OH), 2931, 2882.1(C-H), 1658.4, 1641.3, 1542.1(COO, CONH), 1473.2, 1433.5(C6H4, C4N2H2), 1411.1, 1198.4(SdO), 1322.7(CN), 1053.9(C-O), 930.6, 868, 847.7(C6H4, C4N2H2). Elem. Anal.(%) found (calcd): C 45.13(45.43), N 14.58(14.67), H 6.25(6.22). The average mole ratio of attached GdDTPA and SD to all polymeric repeat units (mol %): GdDTPA 8.17 (as determined by an ICP Atomscan-2000 spectrometer), SD 0.97 (as determined by 1H NMR); UV(H2O, nm): 268. PAEA-Gd-DTPA-SD (M1): IR(KBr, cm-1): 3383.8(NH2), 3069.8, 2977.5, 2932(C-H), 1655.3, 1596.3(COO, CONH), 1437.2(C6H4, C4N2H2), 1405.3, 1177.7(SdO), 1324.1(C-N), 1092.6(C-O), 929.9, 868.3, 800.1(C6H4, C4N2H2). Elem. Anal.(%) found (calcd): C 41.00(41.44), N 20.27(20.49), H 5.74(5.69). The average mole ratio of attached Gd-DTPA and SD to all polymeric repeat units

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(mol %): Gd-DTPA 14.75 (as determined by an ICP Atomscan-2000 spectrometer), SD 3.75 (as determined by 1H NMR); UV(H2O, nm): 301.8. PAEA-Gd-DTPA-SD (M2): IR(KBr, cm-1): 3422.1(NH2), 3068.3, 2927.6(C-H), 1658.0, 1595.2(COO, CONH), 1438.1(C6H4, C4N2H2), 1407.4, 1176.6(SdO), 1326(C-N), 1093.3(C-O), 931.2, 867, 800.7(C6H4, C4N2H2). Elem. Anal.(%) found (calcd): C 37.13(37.86), N 15.35(15.65), H 4.86(4.73). The average mole ratio of attached GdDTPA and SD to all polymeric repeat units (mol %): GdDTPA 45.04 (as determined by an ICP Atomscan-2000 spectrometer), SD 7.54 (as determined by 1H NMR); UV(H2O, nm): 301.8. PABA-Gd-DTPA-SD: IR(KBr, cm-1): 3444.1(NH2), 3079.8, 2932.4(C-H), 1641.5, 1597.8(COO, CONH), 1438.2(C6H4, C4N2H2), 1403.6, 1169(SdO), 1322(C-N), 1092(C-O), 929.9, 866.4, 801.7(C6H4, C4N2H2). Elem. Anal.(%) found (calcd): C 43.69(44.25), N 16.90(17.11), H 6.30(6.24). The average mole ratio of attached GdDTPA and SD to all polymeric repeat units (mol %): GdDTPA 21.67(as determined by an ICP Atomscan-2000 spectrometer), SD 2.21 (as determined by 1H NMR); UV(H2O, nm): 268.5. PAHA-Gd-DTPA-SD: IR(KBr, cm-1): 3434.2(NH2), 3078.9, 2933.7, 2860.8(C-H), 1658.3, 1647.0, 1601, 1542.3(COO, CONH), 1473.2, 1437.3(C6H4, C4N2H2), 1399.3, 1121.4(SdO), 1322.4, 1270.9(C-N), 1092.8(C-O), 930.5, 866.5, 800.8(C6H4, C4N2H2). Elem. Anal.(%) found (calcd): C 51.48(51.78), N 17.56(17.66), H 7.81(7.79). The average mole ratio of attached Gd-DTPA and SD to all polymeric repeat units (mol %): Gd-DTPA 8.86 (as determined by an ICP Atomscan-2000 spectrometer), SD 2.59 (as determined by 1H NMR); UV(H2O, nm): 291. Relaxivity. In the absence of solute-solute interactions, the solvent relaxation rates are linearly dependent on the concentration of the paramagnetic species ([M]); Relaxivity, r1, is defined as the slope of this dependence (2):

(1/T1)obsd ) (1/T1)d + r1[M] where (1/T1)obsd is the observed solvent relaxation rate in the presence of a paramagnetic species, (1/T1)d is the solvent relaxation rate in the absence of a paramagnatic species. In this experiment, the concentrations of the paramagnetic species [Gd3+] were measured by an ICP Atomscan-2000 spectrometer. The solvent longitudinal relaxation time (T1) for gadolinium complexes were carried out on a 10-3 M solution of gadolinium complexes in distilled water. Thus r1 for gadolinium complexes in distilled water could be calculated. MR Imaging. KunMing mice bearing hepatoma (H22) in the right lower limb (weight: 30 ( 2 g) were anesthetized with 10% urethane solution (10 mL/kg), positioned prone and fixed to a polystyrene cradle with adhesive tape to minimize the motion. After performing nonenhanced MR imaging, a solution of PAEA-Gd-DTPA-SD (M1, 0.1 mmol/kg) in 0.9% sodium chloride was injected via the tail vein. Coronal images of the right lower limb were obtained with a T1-weighted spin-echo sequence [Repetition time (TR) 500 ms, echo time (TE) 15 ms, the field of view is 40 mm, with an image matrix of 128×128. Six slices were taken and slice thickness was 2 mm, with a l mm interslice gap.]. Statistical Analysis. Six T1-weighted image slices were taken for the hepatoma in the right lower limb of each mouse. The top and bottom slices were not included in the data analysis to prevent confounding partial volume effects at the edges of the tumors. The average

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Table 1. Mn, Mw, and Polydispersity of Polyaspartamide polymer carriers

Mn (×104)

Mw (×104)

polydispersity

PHEA PAEA PHPA PABA PAHA

2.6 1.2 1.5 2.0 1.8

3.8 1.9 2.4 3.4 2.2

1.46 1.58 1.60 1.70 1.22

Table 2. Relaxivity of Gadolinium Complexesa

a

gadolinium complex

r1 [1/(mmol/L)/s]

Gd-DTPA PHEA-Gd-DTPA-SD (M1) PHEA-Gd-DTPA-SD (M2) PHPA-Gd-DTPA-SD PAEA-Gd-DTPA-SD (M1) PAEA-Gd-DTPA-SD (M2) PABA-Gd-DTPA-SD PAHA-Gd-DTPA-SD

7.0 ( 0.4 24.7 ( 1.2 12.0 ( 0.7 14.8 ( 0.5 14.3 ( 0.7 8.1 ( 0.6 9.9 ( 0.3 9.2 ( 0.2

Temp: 17 °C NMR, frequency: 300 MHz, T1d ) 2.579 ( 0.017

s.

relative signal intensities (pre-injection and postinjection at different times) within the region of interest were normalized to the water label background (RIbackground) at each of the imaging conditions. The percentage of contrast enhancement in the signal from the tumors was calculated according to the following

Figure 1. A is the T1-weighted image of the right lower limb of mice with H22 receiving no MRI contrast agent; B, C, and D are the T1-weighted images of the right lower limb of mice with H22 which received injection with PAEA-Gd-DTPA-SD (0.1 mmol/kg) after 15 min, 30 min, and 60 min, respectively.

enhancement (%) ) 100% (RIpost - RIpre)/(RIpre - RIbackground) RESULTS AND DISCUSSION

A series of water-soluble polyaspartamide ligands PHEA-DTPA-SD, PHPA-DTPA-SD, PAEA-DTPA-SD, PABA-DTPA-SD, and PAHA-DTPA-SD were synthesized and their gadolinium complexes were also prepared. The 1 H NMR and UV spectra showed the characteristic peaks of benzene group, indicating that sulfadiazine was covalently bound to polyaspartamide (Table 1). The IR spectra of the free polyaspartamide ligands showed characteristic absorption peaks of carboxyl with 17151630 cm-1, while these peaks disappeared and strong absorption peaks with 1660-1540 cm-1 were present in IR spectra of their gadolinium complexes. Thus the results exhibited the formation of gadolinium complexes. Moreover, the average mole ratio of attached DTPA to polymeric repeat units increased and the amount of gadolinium ion in the polyaspartamide complexes became larger as the mole ratio of DTPA active ester/(polymeric repeat units) in feed in the reaction process increased. Macromolecular MRI contrast agents usually exhibit more effective relaxation rates than that of the low molecular weight metal complexes alone and improve the relaxivity per gadolinium atom due to a slowly tumbling systems and an increase in rotational correlation time. Table 2 illustrated that polyaspartamide gadolinium chelates containing SD groups possessed higher relaxivities than that of Gd-DTPA. The T1-weight images of hepatoma at various points in time are shown in Figure 1. Compared to those mice receiving no injection of MRI contrast agent, the signal intensities (SI) of the tumors in mice injected with PAEAGd-DTPA-SD (M1, 0.1 mmol/kg) were obviously enhanced, the irradiated portion of the tumors was brighter and the demarcation became clearer during the detection time, while those of the surrounding tissues, such as the muscle in the lower limb, showed a little change. Two hours after injection with PAEA-Gd-DTPA-SD (M1), the signal in hepatoma in mice were still enhanced

Figure 2. Signal enhancement (%) of the tumors in mice at different times after injection with PAEA-Gd-DTPA-SD (M1, 0.1 mmol/kg).

by 138%, indicating it had a prolonged intravascular duration time in tumors of approximately 2 h (Figure 2). Thus the polyaspartamide gadolinium chelates containing SD groups could provide the prolonged enhancement of the tumors (Figure 2). In summary, polyaspartamide gadolinium complexes containing sulfadiazine groups possess higher relaxation effectiveness than Gd-DTPA. MR imaging showed that PAEA-Gd-DTPA-SD (M1) greatly enhanced the contrast of MR images of hepatoma in the lower limb of mice and provided prolonged intravascular duration. Thus the polyaspartamide gadolinium chelate is expected to be used as the potential macromolecular MRI contrast agents for hepatoma in mice. ACKNOWLEDGMENT

We thank the QUT’s Postdoctoral Research Fellowship Scheme and the Chinese National Natural Science Foundation (No. 29874028) for their financial support. LITERATURE CITED (1) Lauterbur, P. C. (1973) Image formation by induced local interactions: examples employing nuclear magnetic resonance. Nature 242, 190-191.

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