Experimental Study Of Magnetic Resonance Imaging With Folate Receptor Targeted Contrast Agent

PartⅠPreparation and evaluation of BSA-Gd-DTPA and folate-BSA-Gd-DTPAPurpose: To synthesize BSA-Gd-DTPA and folate-BSA-Gd-DTPA, determine the pharmaceutical properties and study in vitro magnetic resonance imaging and cytochemical fluorescence imaging of folate receptor. Methods: DTPA was transformed to cDTPAa and conjugated to bovine serum albumin (BSA) at the molar ratio of 50:1．GdCl was reacted with BSA-DTPA and the unreacted Gd ion was dialyzed and separated from BSA-Gd-DTPA. Using EDC as linker, folate was covalently coupled to BSA-Gd-DTPA at the molar ratio of 10:1．The Gd ion conjugation rate of BSA-Gd-DTPA was measured using ICP-AES and the folate in folate-BSA-Gd-DTPA was detected by spectrophotometer. MR T1W scan was performed to qualitatively determine the uptake of folate-BSA-Gd-DTPA, BSA-Gd-DTPA and Gd-DTPA by the human hepatocelluar carcinoma cell line SMMC-7721 at different concentration. BSA and folate-BSA were labeled with fluorescein isothiocyanate (FITC) and fluorescent staining was performed to determine the uptake of FITC-BSA and FITC-folate-BSA by SMMC-7721．Results: BSA-Gd-DTPA and folate-BSA-Gd-DTPA was successfully synthesized. The Gd ion in BSA-Gd-DTPA was 11．01mg, the conjugation rate was 70% and the loading capacity was 8．47%. The folate in folate-BSA-Gd-DTPA was 63．27mg. The T1W signal of folate-BSA-Gd-DTPA binding to SMMC-7721 was higher than that of BSA-Gd-DTPA binding and Gd-DTPA binding, and the T1W signal of folate-BSA-Gd-DTPA binding decreased with the concentration of SMMC-7721．FITC-folate-BSA was observed to bind to the cell surface of SMMC-7721, while little fluorescent binding was found with FITC-BSA. Conclusions: Folate-BSA-Gd-DTPA could be used as the potential MR contrast agent targeted to the folate receptor.PartⅡMagnetic resonance imaging of folate-BSA-Gd-DTPA in normal micePurpose: To study in vivo MR imaging, distribution and excretion of folate-BSA-Gd-DTPA in normal mice. Methods: 30 Balb/c mice were radomly divided into three groups and administered with three contrast agents: Gd-DTPA, BSA-Gd-DTPA and folate-BSA-Gd-DTPA. A 1．5 Tesla Siemens MR scanner and the head coil were used. The plain T2W FSE and T1W SE sequences were performed. The repeated T1W SE sequences were then performed 15min, 30min, 1h, 2h, 4h, 6h, 8h, 12h, 18h and 24h after the intravenous injection of Gd-DTPA, BSA-Gd-DTPA and folate-BSA-Gd-DTPA. The signal intensity (SI) of the inferior vena cana, liver, muscle and renal cortex in the pre-and post-contrast T1WI was measured using a circular region of interesting with 5 pixels and the relative enhancement (RE) was calculated as (SI-SI) / SI×100%. The appearance of the contrast agent in the gallbladder was observed. Results: The RE of the inferior vena cana was statistically different at the time point of 15min, 30min, 1h, 2h, 4h, 6h, 8h and 12h among three groups, the RE of the liver at the time point of 15min, 30min, 1h, 2h, 4h, 6h, 8h, 12h, 18h and 24h, the RE of the muscle at the time point of 30min and 1h, and the RE of the renal cortex at the time point of 15min, 30min, 1h, 2h, 4h, 6h, 8h, 12h, 18h and 24h. The RE of the renal cortex after folate-BSA-Gd-DTPA enhancement was higher than the RE after BSA-Gd-DTPA enhancement at the time point of 4h, 6h, 8h, 12h, 18h and 24h. 8 mice administered with BSA-Gd-DTPA and 9 mice with folate-BSA-Gd-DTPA had high signal intensity caused by contrast agent in the gallbladder at the time point of 18h and 24h, while all 10 mice with Gd-DTPA did not have the contrast agent in the gallbladder. Conclusions: BSA-Gd-DTPA and folate-BSA-Gd-DTPA could result in more and persistent enhancement of the blood, liver and renal cortex and excreted partially through the biliary route. Folate-BSA-Gd-DTPA could target to the renal cortex and be imaged by MR.PartⅢMagnetic resonance imaging of folate-BSA-Gd-DTPA with orthotopic transplantation of human hepatocellular carcinoma in nude micePurpose: To study in vivo MR imaging of folate-BSA-Gd-DTPA with orthotopic transplantation of human hepatocellular carcinoma in nude mice. Methods: The human hepatocellular carcinoma cell line SMMC-7721 was cultured and 0．2 ml suspended cells with the concentration of 1．0×10 /ml were inoculated into the subcutaneous tissue of the dorsal subscapular region of 4 athymic nude mice. After the subcutaneous tumor developed into the bulky mass, the tumor was excised and cut into the fragments with the diameter of 0．5 mm. The fragments were then implanted through the envelope into the liver parenchyma of 20 nude mice. Three to four weeks after the liver transplantation, 20 nude mice were randomly divided into two groups and administered with BSA-Gd-DTPA and folate-BSA-Gd-DTPA. MR T2W FSE sequence and T1 W SE sequences before and after the intravenous injection of two contrast agents were performed. The scan parameters and the time points were the same as partⅡ. The T1 W SI of the tumor, liver and renal cortex was measured and the RE was calculated. The value of tumor-to-liver contrast to noise ratio (CNR), which corresponds to the conspicuity score, was obtained with the fomular of (SI-SI) / background noise and the relation of CNR to the time point was observed. The MR imaging features of the tumor after BSA-Gd-DTPA and folate-BSA-Gd-DTPA enhancement were analyzed and compared with the pathologic results. Results: The orthotopic transplantation model of the human hepatocellular carcinoma was successfully obtained in 20 nude mice. All the hepatocellular carcinoma were detected by MR and the range of the tumor size was from 7．4mm×6．8mm to 14．8mm×12．1mm. The RE of the tumor after folate-BSA-Gd-DTPA enhancement was higher than the RE after BSA-Gd-DTPA enhancement at the time point of 2h, 4h, 6h, 8h, 12h, 18h and 24h. The CNR of tumor-to-liver reached peak at the time point of 8h. The tumor is exhibited as hyper-intensity compared with the liver parenchyma after folate-BSA-Gd-DTPA enhancement, while exhibited as hypo-intensity after BSA-Gd-DTPA enhancement. The enhancement mode of the tumor correlated with the size and pathological structure. Conclusions: Folate-BSA-Gd-DTPA could target to the hepatocellular carcinoma and be imaged by MR. Folate-BSA-Gd-DTPA could be of qualitative value in the diagnosis of hepatocellular carcinoma.