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Lactoferrin-conjugated Superparamagnetic Iron Oxide Nanoparticles as a Specific MRI Contrast Agent for Detection of Brain Glioma in Rats

Author: XieHui
Tutor: YangXiangLiang；XuHaiBo
School: Huazhong University of Science and Technology
Course: Biopharmaceutical engineering
Keywords: Lactoferrin Low-density lipoprotein receptor-related protein 1 Glioma Superparamagnetic iron oxide nanoparticles Contrast agent Magnetic resonance imaging
CLC: R739.4
Type: PhD thesis
Year: 2011
Downloads: 50
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Abstract

Invasive glioma is the most common primary brain tumor of adults. The proportion of all primary brain tumors, 40%, and accounted for 78% of the malignant brain tumor. The paramagnetic gadolinium contrast agent for magnetic resonance imaging (MRI) examination is the most commonly used diagnostic methods of the glioma. Gadolinium contrast agent, however, there are many problems in the clinical application of diffusion imaging easy to surrounding normal tissue cause the imaging site boundary is fuzzy, short imaging time is not conducive to the postoperative diagnosis of the tumor, In addition, studies in recent years found in the liver and kidney transplant patients with nephrogenic fibrosis associated with the use of gadolinium. Compared with gadolinium contrast agents, superparamagnetic iron oxide contrast agent with strong magnetic properties and good biocompatibility, but it is also a non-specific contrast agent, can not be targeted to specific organizations. Although there have been reported on targeting glioma MRI contrast agent, but they are still in the practical application, there are many restrictions. Family of lactoferrin (Lf) transferrin (Tf) with a positive charge under physiological conditions, the low plasma concentration of its receptor (LRP1) in the blood-brain barrier (BBB) ??and some glioma (U87 brain gliomas) were expressed. Therefore, Lf suitable as ligands targeting glioma. In this study, using EDC coupling method, the hyper-paramagnetic iron oxide nanoparticles (SPIONs) Lf coupled prepared the lactoferrin conjugated superparamagnetic iron oxide nanoparticles (Lf-SPIONs). Lf-SPIONs on this basis, in terms of the physicochemical properties and the magnetic can examine the possibility as a MRI contrast agent; Lf-SPIONs investigated from two aspects of the in vitro imaging and in vivo imaging of rat orthotopic glioma imaging LRP1 (low-density lipoprotein receptor-related protein 1, LRP1) expression in glioma characteristics; preliminary investigation receptor expression, and to explore the mechanism of Lf-SPIONs selective accumulation of glioma; from cells Lf-SPIONs bio-security and animal two levels investigated. The major work completed in the study include: (1) investigated the the LRP1 expression of glioma. RT-PCR and Western Blotting results show that human glioma cell line SHG-44 and U251 and rat glioma cells C6 has LRP1 expression of normal brain tissue in rats, glioma tissues and paracancerous of the organization have LRP1 expression and LRP1 expression of brain tumor tissue than normal brain tissue and cancer tissue. Immunohistochemical results also confirmed that LRP1 expression in glioma tissues. (2) using EDC coupling prepared Lf-SPIONs. By polyacrylamide gel electrophoresis (PAGE) and Fourier transform infrared spectroscopy (FT-IR) method of confirmatory Lf and the SPIONs the coupling. Particle size of SPIONs and Lf-SPIONs were 9.2 ± 0.4 nm and 13.6 ± 0.7 nm hydrodynamic radius were 62.0 ± 12.1 nm and 74.8 ± 11.5 nm, the intensity of the magnetic saturation were 78 emu / g Fe and 51 emu / g Fe, relaxation rate were 131.4 S ^{-1 mM ^{-1 and 75.6 S ^{-1 mM ^{-1 . Bradford method to determine the SPIONs with LF coupling ratio of 1 mg: 0.1 mg (SPIONs: LF). (3) the use of C6 cells (LRP1 positive) and the ECV304 cell (LRP1 negative) effects of glioma cell uptake of Lf-SPIONs. The results showed that the two types of cells can ingest SPIONs and Lf-SPIONs C6 cells Lf-SPIONs intake was significantly higher than SPIONs; ECV304 cells was no significant difference between the intake. LRP1 positive C6 glioma cells selective uptake Lf-SPIONs, this selective uptake may be by Lf mediated endocytosis completed within cells enriched. Cell electron microscopy and Prussian blue staining results showed that the cellular uptake of SPIONs and Lf-SPIONs are located in the cytoplasm. (4) MRI investigated Lf-SPIONs glioma imaging characteristics. The results showed that Lf-SPIONs can specifically targeted the rat glioma imaging, and imaging for more than 48 h. By intracranial inoculation method of rat C6 glioma cells, rat glioma model. Glioma MRI enhancement effect found by comparing SPIONs and Lf-SPIONs tail vein injection of Lf-SPIONs (12 mg / kg) 2 h after the MR image signal of the tumor-bearing rats with tumors began to decrease, and the signal is reduced with the the time gradually expand the MR image signal is reduced by 74.81%. In tumor-bearing rats injected with the same dose of SPIONs, MR image does not appear gray-scale changes in the visible, only 10.05% of the maximum signal reduce, even if the injection of a large dose of SPIONs (30 - 50 mg / kg) 48 h after the tumor area yet to see any change in signal intensity. But this time, the liver and kidneys were apparent signal change SPIONs has entered the circulatory system of the rat, but not into the brain. In addition, normal rats injected Lf-SPIONs (12 mg / kg), brain MR image signal intensity change does not appear, Lf-SPIONs can not cross the BBB into the brain. Tumor tissue Prussian blue staining showed injection Lf-SPIONs rat tumor tissue has accumulated a large number of iron particles and is located within the tumor cells, thus confirming the Lf-SPIONs can reach tumor tissue. Injection SPIONs rat tumor, only in the tumor edge blood vessels memory in a small amount of iron particles, not enough to cause visible on the MR image signal change. Atomic absorption results Table the Ming He tumor rat brain tumor accumulation of large amounts of iron, further confirmed the Lf-SPIONs targeted selective glioma. (5) Lf-SPIONs the security in vitro and in vivo security in a preliminary investigation. Thiazolyl blue (MTT) assay results 0 - 100μg/mL concentration range, the growth and proliferation of SPIONs and Lf-SPIONs three normal cells (HEK293, HL-7702 and ECV304 cells) had no significant effect. Vivo safety experiment results show that, after intravenous injection of SPIONs and Lf-SPIONs, rat liver and kidney function indicators (including aspartate aminotransferase (AST), alanine aminotransferase (ALT), total protein (TP), albumin (ALB), blood urea nitrogen (BUN), creatinine (CREA) and serum total cholesterol (CHOL)) and blood function indicators (including white blood cell count (WBC), red blood cell count (RBC) and hemoglobin concentration (HGB)) were not appear abnormal. In addition, histopathological examination of the results of the various organs (heart, liver, spleen, lung, kidney and brain tissue) SPIONs and Lf-SPIONs did not cause structural abnormalities of the organ. These results were confirmed the SPIONs and LF-the SPIONs no toxicity, and is a good safety of the MRI contrast agent. In summary, Lf-SPIONs Lf targeting of brain tumors and SPIONs as MRI contrast agents organically combine excellent image capabilities, accurate positioning solution preoperative tumor tissue for surgery Radiation therapy to develop programs. Also on postoperative residual tiny stove, recurrent tumors early qualitative, accurate positioning and comprehensive treatment guide. In addition, Lf-SPIONs has good bio-security, and there are broad prospects for clinical application in targeted diagnosis of glioma.}}}}

Lactoferrin-conjugated Superparamagnetic Iron Oxide Nanoparticles as a Specific MRI Contrast Agent for Detection of Brain Glioma in Rats

Abstract

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