The Synthesis Of Tumor-Targeted Magnetic Resonance Imaging Contrast Agents And Their Application In Early Diagnosis Of Cancer

Magnetic resonance imaging (MRI) is currently one of the most useful noninvasive techniques in clinic for assessing anatomy and function of tissues and organs. The novel applications such as imaging of gene expression and real-time evaluation for drug delivery in vivo are actively studied. This technique is characterized by its excellent temporal and spatial resolution, its non-exposure to radiation and long effective imaging windows. However, MRI is less sensitive than nuclear medicine when used to monitor small tissue lesions, molecular activity, or cellular activities. With the appilication of paramagnetic contrast agents such as chelated Gd-diethylenetriaminepentaacetate (Gd-DTPA) or Gd-tetraazacyclododecanetetraacetic acid (Gd-DOTA), the imaging contrast and sensitivity can be increased by shortening the T1-relaxation time of water protons, and thus the tumor image appears brighter in the T1-weighted image. Several kinds of low molecular MRI contrast agents based on Gd-DTPA and Gd-DOTA have been approved by American Food and Drug Administration and widely used in clinical diagnosis of tumors. However, these low molecular weight contrast agents have serious drawbacks which limit their application for MRI contrast agents, such as short half-life in blood, low relaxivity rate, and lack of specificity to target organs and tissues for early diagnosis of tumor. To overcome the above disadvantages and achieve the early diagnosis of tumors, macromolecular MRI contrast agents have been developed by the conjugation of DTPA or other chelating units and polymer carriers. Polymers such as poly (ethylene glycol) (PEG), dextran, poly (L-lysine), poly (glutamic acid), poly [N-(2-hydroxypropyl) methacrylamide],α,β-poly [N-(2-aminoethyl) aspartamide], disulfide-based biodegradable synthetic polymers, dendrimer and polymeric micelles have been investigated as the carriers of gadolinium complexes. Macromolecular MRI contrast agents showed increased contrast, sensitivity, diagnostic imaging time, and specificity to tumor tissues for early dignosis after conjugated with targeting moieties such as folate, antibody or peptide.In this study, we prepared two kinds of MRI contrast agents. One is FA-PEG-PAMAM-Gd nanoparticles as potential lung cancer-targeted macromolecular MRI contrast agent. Another one is polymer-gadolinium complex nanomicelle of Pep-PEG-g-PAA-(DTTA-Gd) as MRI contrast agent for early diagnosis of liver cancer. FA-PEG-PAMAM-Gd MRI contrast agent was prepared without using low molecular ligands for the reason of increased cost in clinical applications. In particular, poly (amidoamine) (PAMAM) dendrimer with 32 carboxylic groups was modified with folate-conjugated poly (ethyleneglycol) amine (FA-PEG-NH2, Mw:1 k,2 k and 4 kDa). FA-PEG-PAMAM-Gd macromolecular MRI contrast agents were prepared by the complex reaction between the carboxylic groups in PAMAM and GdCl3. The structure of FA-PEG-PAMAM-COOH was confirmed by nuclear magnetic resonance (1H NMR), Fourier transform infrared (FT IR) spectra and electrospray ionization mass spectra (ESI-MS). The mass percentage content of Gd (Ⅲ) in FA-PEG-PAMAM-Gd was measured by inductively coupled plasma-atomic emission spectrometer (ICP-AES). The sizes of these nanoparticles were about 70 nm measured by transmission electron microscopy (TEM), suggesting their passive targeting potential to tumor tissue. In comparison with small molecular available in clinc, Gadopentetate dimeglumine, FA-PEG-PAMAM-Gd showed comparable cytotoxicity and higher relaxation rate. The prepared samples, with folic acid as the targeting group, were evaluated for their potentiality as tumor-targeting MRI contrast agents. A Siemens Tim Trio human MRI scanner at 3 T was used to test the concentration detection limits in vitro, contrast-enhanced MR imaging in the heart, kidney, and liver of mice and the metabolism action of FA-PEG-PAMAM-Gd. A transmission electron microscopy was used to determine the targeting to tumor cells. The toxicity was also assayed to evaluate the biocompatibility. The minimal detectable concentration for FA-PEG4k-PAMAM-Gd (PEG:4000 Da) was more than 15-fold lower than that for the commercially available contrast agent Gadopentetate dimeglumine. MRI images showed a gradual and prolonged tumor signal enhancement for FA-PEG4k-PAMAM-Gd. Millimeter-sized (-5 mm) tumors were well-visualized using the same agent. Our results indicate that the dendritic contrast agents, FA-PEG-PAMAM-Gd, have bonded highly to both KB and A549 cell lines, and the molecule targeting can be monitored by them in future.However, the greatest risk from MRI contrast agents is the release of gadolinium from the chelate, which can lead to nephrogenic systemic fibrosis or acute metal toxicity. To decrease the toxicity of contrast agents, very recently, much attention was focused on polymeric nanomicelle MRI contrast agents. The polymeric nanomicelle MRI contrast agents can be dissociated into copolymer chains and excreted from kidney over a period, and so the toxicity could be reduced. In addition, polymeric nanomicelle MRI contrast agents exhibit a preferential pharmacokinetic profile during targeting of tumors, and their high structural stability in the bloodstream.Another polymeric nanomicelle MRI contrast agent, Poly (ethylene glycol)-graft-α,β-poly (aspartic acid) derivatives (MAL-PEG-g-PAA-N3), were synthesized by sequential ring-opening reaction of polysuccinimide (PSI) with MAL-PEG-NH2 (PEG:2000 Da, MAL: N-(6-Maleimidocaproyloxy) succinimide) and 1-azido-3-aminopropane, respectively. Then N2-(hex-5-yne)-diethylenetriamine-tetra-t-butylacetate (DTTA-der) was conjugated to MAL-PEG-g-PAA-N3 by click cycloaddition. After deprotection of carboxylic groups, MAL-PEG-g-PAA-DTTA-COOH macromolecular ligands were obtained, which were then conjugated with targeting groups of peptides. Pep-PEG-g-PAA-(DTTA-Gd) (Pep:peptide) complex nanomicelles were fabricated from Pep-PEG-g-PAA-DTTA-COOH and gadolinium chloride. The formation of nanomicelles was confirmed by fluorescence spectrophotoscopy and particle size measurements. All the nanomicelles showed spherical shapes with core-shell structures and narrow size distributions. Their sizes ranged from 50 to 80 nm, suggesting their passive targeting potential to tumor tissue. With the increase of graft degree (GD) of PEG, the sizes of Pep-PEG-g-PAA-(DTTA-Gd) nanomicelles showed a tendency to increase. Compared with gadopentetate dimeglumine (Gd-DTPA), Pep-PEG-g-PAA-(DTTA-Gd) nanomicelles showed decreased cytotoxicity, good biocompatibility and enhanced T1-weighted signal intensity, especially at low concentration of gadolinium (Ⅲ). Tumors with diameter of 5 mm were well-visualized using this contrast agent suggesting their great potentiality as MRI contrast agents.