Preparation And Imaging Of Folate-targeted Nano-magnetic Resonance Contrast Agent: A Preliminary Study

Background Cancer is a kind of common disease that is harmful to human health. New cases of cancer worldwide reported by the World Health Organization each year is more than 10 million including 5.3 million men and 4.7 million women. The number of deaths for new cases is more than 7 million. The death caused by cancer is up to 12%of the total deaths. By 2020, new cases of cancers is surposed to reach 15 million worldwide per year. The total number of cancer patients will grow by 73%in developing countries, and 29%in developed countries. Cancer is the one of the leading causes of death in China. By the end of 2008, China's new cases of cancer each year has been 2.2 million and the number of cancer deaths is 1.6 million. In the past 30 years, the incidence of cancer among residents has had an increase of 37%of men and 44.76%of women who were in the best labor state at the age from 20 to 60 accounted for 40%. Lung cancer and breast cancer ranked the first among the male and female, respectively. The top five cancer with highest mortality are:lung cancer, carcinoma of stomach, liver cancer, colorectal cancer and breast cancer. Targeted magnetic resonance contrast agent can not only improve the diagnostic accuracy, but also reflect the biological characteristics of some diseases and evaluate the treatment efficiency. They have excellent perspective in clinical application and have become one of the hotspots in molecular imaging studies. It is shown in Molecular biology studies that the surface of tumor cells or tumor-associated blood vessels have a series of receptors over-expressed in tumor tissue. The receptors can bind with the ligands or the corresponding ligands specifically. Receptor and its ligand's binding have the feature of specificity, selectivity, saturation, strong affinity, good biological effects, and so on. It gives a new targeted approach to the diagnosis. Folate receptor (folate receptor, FR)over-expresses in many human tumors, such as breast cancer, ovarian cancer, endometrial cancer, lung cancer, kidney cancer, colon cacer, cerebroma and the cancer origination from bone marrow hematopoietic cells and sometimes it is 20-300 times higher than that of normal tissue. This over-expression of folate eceptors (FR) on cancer tissues can be exploited to target folate-linked imaging and therapeutic agents specifically to FR-expressing tumors, thereby avoiding being uptaken by most normal tissues that express little if any FR. Folate conjugates are taken up by cancer cells via an FR-mediated endocytic pathway so as to diagnose and treat.Objective To prepare the particle with appropriate size to connect folic acid nanoparticle contrast agent. To explore the feasibility of the macromolecular contrast agent for tumor targeting and the characteristics of imaging in vivo with FR-positive tumor cells in nude mice model.Methodsâ… Preparation of MRI the targeted Contrast Agent and Characterization in vitroThree different polyethylene glycol diamine (PEG) with molecular weight of 1000,2000 and 4000 respectivly were synthesized with folic acid to produce three polyethylene glycol diamine with different molecular weight combined with folic acid residues respectively. Compounded with small molecule ligand diethylenetriaminepentaacetic pentaacetic acid (DTPA) to prepare PAMAM-DTPA-PEG-FA/Gd, with different molecular weight. They are PEG 1000, PEG2000, PEG4000.To apply inductively coupled plasma atomic emission spectrometer to measure Gd content and to assess its activity with cell folate receptor competitive binding assay.â…¡The toxicity of the contrast agents in vitro and relaxativityTo evaluate and compare the influence on KB cell survival rate by newly synthesized contrast agent (PEG1000, PEG2000, PEG4000) and conventional contrast agent (Gd-DTPA) in vitro with MTT assay. To evaluate the effectiveness of the various contrast agents relaxativity with T1 MAPPING assay.â…¢Specific and non-specific magnetic resonance contrast agent tumor xenografts Magnetic resonanceimaging1. Study on MRI targeted contrast agent (PEG4000) targeting in nude mice bearing human oral epithelial carcinomaThe KB human oral epithelial cell were inoculated subcutaneously in nude mice to produce human oral epithelial carcinoma model. To administer targeted MRI contrast agent PEG4000 by the rat tail vein, and to observe signal value changes of tumor, liver, and leg muscles of the model in T1WI respectively before and after the administration at different time points.2. To compare the influence by MRI targeted contrast agent (PEG4000), non-targeted groups of PEG2000 with conventional contrast agent (Gd-DTPA) respectively in nude mice bearing human oral epithelial carcinoma-control.Nude mice(n=12)bearing human oral epithelial carcinoma were randomly divided into 3 groups, including PEG4000 contrast agent group(n= 4), non-targeting of the PEG2000 group (n= 4) and conventional contrast medium group (n= 4), respectively. After the injection of targeted contrast agent PEG4000, non-targeting of PEG2000 and Gd-DTPA respctilvely through the rat tail vein, T1WI signal value changes of tumors and muscle before and after injection at different time points were observed and compared.Results 1. Folic acid targeting moiety with polymer macromolecules PEG 1000, PEG2000, PEG4000 were successfully prepared. the polymer has high purity and high affinity with folate receptor; meanwhile, they also carry with a high concentration of Gd3+ions (per molecule containing 32-36 Gd3+);2.0f the three newly synthesized kinds of contrast agents, PEG4000 group has the highest cell survival rate with minimal toxicity. The relaxativity of three kinds of macromolecular chelate are equivalent and approximately three times as that of conventional contrast agent.3. In MRI study on the targeted nude mice bearing KB cell tumor, the reagents PEG4000 has the accumulation trend of localizing to the tumor. The signal value peak merges in about 3-4 hours after injection and the maximum enhancement ratio is 116.0%; The enhancement effect in the liver is also apparent and the enhanced peak occurs in about 0.5-2 hours, followed by the slow downward trend.4.To compare non-targeting of the PEG2000 with conventional contrast agent (Gd-DTPA) contrast, (1) the PEG4000 contrast agent has significant effect on tumor targeting. There is statistical difference in the signal values of the PEG4000 group at different time points; the enhanced peak occurs in about 3-4 hours after injection; no significant difference of the signal values of non-targeting of the PEG2000 groups at different time points; the enhanced peak occurs in about 2-3 hours after injection; the signal value of the muscles in the PEG4000 group and non-targeting of the PEG2000 group are overall modest at different scan times. (2) there was significant statistical difference in the signal values of the conventional contrast agent (Gd-DTPA) group at different time points; the enhanced peak occurs in about 0.5 hours after injection; in the control group, the signal value peak of the muscles occurs in 0.5 hours after the injection.Conlusions The highly binding of folic acid with targeted group with PEG4000 helps to intensify the effect in MR enhancement in nude mice model, so as to fufill for target imaging and diagnosis in MR...