| Glioma is the most aggressive brain tumor in humans with a survival rate of only 5% after five years. Magnetic resonance imaging (MRI) with gadolinium-based contrast agent is currently the gold standard for the detection of brain tumor. However, the gadolinium-based contrast agent has poor target-specific biodistribution, rapid exertion and undesired side-effects in patients. Therefore, superparamagnetic iron oxide nanoparticles (SPIONs), which have high sensitivity, nontoxicicity and excellent biocompatibility, has been investigated in vivo in brain tumors. Since they have no sensitivity towards tissues and are unable to discriminate between malignant and non-malignant tumors, the conjugation of specific ligands with SPIONs has been employed in MR visualization to improve the tissue contrast selectivity. Lactoferrin (Lf), which belongs to the transferrin (Tf) family, has much lower plasma concentration under physiological conditions. Furthermore, it was reported that Lf receptors (LRP1) expressed on the glioblastomas (U87 gliomas) and blood brain barrier (BBB). All of these make Lf a better ligand to target brain glioma. In this study, Lf was conjugated to SPIONs using the EDC method. The level of LRP1 expressing on gliomas, the ability of Lf-SPIONs as an MRI contrast agent, the specificity of Lf-SPIONs to brain gliomas and the biocompatibility of Lf-SPIONs have been evaluated both in vitro and in vivo. The main results are:(1) RT-PCR and Western blotting analysis indicated that LRP1 was expressed in various glioma cell lines and glioma tissues. Although LRP1 was expressed in normal tissues, adjacent tumor tissues and glioma tissues, the level of LRP1 in tumor tissues was much higher (p < 0.05). Immunohistochemical analysis also verified that LRP1 was expressed in brain glioma tissues.(2) The results of polyacrylamide gel electrophoresis (PAGE) and Fourier transform infrared spectroscopy (FT-IR) proved that Lf conjugated successfully to the SPIONs. The conjugation amount of Lf was 0.1 mg to 1 mg SPIONs, which was determined by Bradford method. The overall size increased from 9.2±0.4 nm (bare SPIONs) to 13.6±0.7 nm (Lf-SPIONs) on transmission electron microscope (TEM) images. The corresponding hydrodynamic diameters of both nanoparticles were 62.0±12.1 nm (bare SPIONs) and 74.8±11.5 nm (Lf-SPIONs) respectively. The saturation magnetization of SPIONs and Lf-SPIONs measured by vibrating sample magnetometer (VSM) were 78 emu/g Fe and 51 emu/g Fe at room temperature. The MRI results suggested the R2 relaxivity coefficient value (r2) of Lf-SPIONs was 75.6 mM-1 S-1, resulting in better contrast effects.(3) The specificity of Lf-SPIONs was studied in vitro. Both SPIONs and Lf-SPIONs could be uptaked by C6 cells (LRP1 positive). Compared to the group treated with SPIONs, the group treated with Lf-SPIONs had a higher Fe content. Besides, after treated with Lf-SPIONs, the signal intensity of C6 cells was decreased significantly than that of SPIONs group. All these results suggested that Lf could possibly enhance the cellular uptake with SPIONs in glioma cells. Meanwhile, SPIONs and Lf-SPIONs also could be uptake by ECV304 cells (LRP1 negative). However, the results showed no significant difference between the SPIONs group and the Lf-SPIONs group. Consistent with the cell uptake studies above, TEM images indicated that Lf-SPIONs were accumulated in the cytoplasm of C6 cells.(4) The specificity of Lf-SPIONs was studied either in vivo. On MR images, high contrast of brain tissues was observed with Lf-SPIONs (12 mg/kg) from 2 to 48 hours, while MR imaging with SPIONs (12 mg/kg) showed no visible change over the same period, even at high dose (30 - 50 mg/kg). The significant decrease of MR signal intensity was about 36 - 74% at the tumor site post contrast compared with the pre-contrasted tumor. Consistent with the findings of MR imaging, both the results of Prussian blue staining and atomic absorption Spectroscopy verified large amount of Lf-SPIONs accumulated into tumor tissues whereas rare SPIONs were accumulated. Besides, no difference was observed in the normal brain of rat treated with Lf-SPIONs (12 mg/kg) in spite of the report that Lf could cross the BBB.(5) The biocompatibility of Lf-SPIONs was evaluated in vitro and in vivo. It was found from MTT assay that the growth of all tested normal cells was not affected even exposing to the Lf-SPIONs with high concentration and no noticeable cytotoxic effect was observed. Furthermore, the biochemical analysis (AST, ALT, TP, ALB, BUN, CREA and CHOL) and the blood analysis (WBC, RBC and HGB) verified that Lf-SPIONs didn't affect the important biological function of rats. Histopathological results showed that after injection with Lf-SPIONs, all the detected organs (heart, liver, spleen, lung, kidney and brain) had normal structures and no noticeable cytotoxic effect was observed even at 21 days.In summary, we have identified and developed a contrast agent (Lf-SPIONs), which could be bound to brain glioma with high selectivity and sensitivity. With the greater accumulation in glioma and improving high contrast between the tumor and surrounding normal brain tissue over a period of 48 h, such glioma-targeted Lf-SPIONs would be useful in both preoperative and postoperative for delineating tumor. Besides brain glioma, Lf-SPIONs may have great potential to be employed in diagnosis of other carcinogenic tumors.
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