Biological Effects And MR Signal Detection Of Different Materials Coated SPIOs On Macrophages

Introduction:The phagocytic cells including T cells, B cells and the most macrophages play a very important role in the progression and recovery of the most inflammatory reaction, some cardiovascular system's diseases and nervous system's degenerative diseases. Usually we evaluate these diseases' inflammatory reaction with histological examination. But such methods can only observe the existence of inflammatory lesions in static condition, can't observe the process of inflammatory cells migrating to the lesion dynamically. So the process of progression and recovery of these diseases can be appreciated if we can track the phagocytic cells' aggregation and inversion in the diseased regions dynamically and non-invasively. As the fast development of molecular imaging, a promising new direction for contrast- enhanced magnetic resonance (MR) imaging involves tracking the migration and biodistribution of SPIO-labeled phagocytic cells in vivo. When the SPIOs enter into the blood circulation, they can be uptake by endothelial system and the phagocytes in the blood. When the SPIOs are uptake by cells, they can't come back to the blood any more. Then the phagocytes are labeled with SPIOs. The labeled cells can move and deposit in the place of inflammation. Then the T₂ relaxation time of the place with inflammation reaction is shorten by labeled cells which caused the signal decrease.SPIOs typically consist of two components, an iron oxide core and a hydrophilic coating. SPIOs sizes range greatly from 2 to 150nm, and there are many kinds of coating materials. The existence of coating materials can elevate the stability and biocompatibility of SPIOs. And the SPIOs become targeted probes after the farther modification. There have been many studies that labeling the stimulated or unstimulated phagocytes using SPIOs and evaluating the inflammation reaction in vivo. And the huge predominance has been proved by the studies using SPIOs. But the results of the studies were poor compareble due to the different coating material and diameter of SPIOs. If we can find the optimal SPIOs configuration which not only have a high labeling efficiency but also have a high biocompatibility, it is very important for the future studies. In this present study, in order to understand which SPIOs is suitable to labeling phagocytes, we labeled the macrophages with citric-acid and dextran coated SPIOs and compared the differences of labeling efficiency, MR signal and cell functions in vitro.Materials and Methods:According to the coprecipitation methods, we synthesized the citric-acid and dextran coated SPIO. Freshly isolated 6 weeks old female mouse peritoneal macrophages were labeled with SPIOs of two materials, citric-acid and dextran, for 1 hour in culture. The Prussian blue staining was used to evaluate the labeling efficiency of two coating materials. The Calcein-AM was used to evaluate the cell viability labeled with SPIOs of two materials (0.1, 0.25, 0.5, 0.75, 1.0mg/ml). When the macrophages were incubated with SPIOs (0.5mg/ml) of two materials, the migration capability was observed by cell scratch test which was evaluated by imageJ. Levels of proinflammatory cytokine, interleukin-6, was measured with enzyme-linked immunosorbent assay 24 hours after labeling with the SPIOs (0.5mg/ml) of two materials for 1 hour. After labeling the cells with two materials coated SPIOs (0.1, 0.25, 0.5, 0.75, 1.0mg/ml) for 1 hour, the cells were fixed in 0.5% agar gel. Then the fixed cells were scanned by PHILIPS 3.0T MR to evaluate the relaxation time of different concentration. The effect of increasing iron concentrations on relaxation time and cell viability were evaluated with two-way analysis of variance, followed by a multiple-comparison procedure. Cell migration and level of IL6 were evaluated with student t test.Results:The citric-acid and dextran coated SPIOs synthesized by co-precipitation were brownish-black colloid fluids. The SPIOs' diameter of hydration was about 100nm and 150nm, and the distribution of nanoparticles size was very good. And the SPIOs were stored on 4°C. The Prussian staining results showed that the macrophages without the SPIOs were homogeneous red, and the cells after intracellular SPIOs had many blue grains in the cells. Obvious, there were more grains in cells which cultured with citric-acid coated SPIOs than dextran coated SPIOs. The cell viability results showed that the both groups' viability were decreased by the increasing of SPIOs concentration. We also compared the viability between the two coating materials, and the results showed that there were no statistical different between two coating materials in the same concentration. The migration capability results showed that there was no statistical difference between the experimental groups and the control groups. The levels of proinflammatory cytokines results showed that there was no statistical difference between experimental groups and control groups with dextran coated SPIOs, and the level of citric-acid coated SPIO was high. In the T2 weighted mapping images, the citric-acid coated SPIO groups' MR signal decrease obvious. However the dextran coated groups' MR signal change was not notable affected even incubated with the highest concentration.Conclusion:In vitro labeling of macrophages was more effective by citric-acid coated SPIOs than dextran coated SPIOs. The macrophages which incubated with citric-acid coated SPIOs resulted in efficient labeling detectable on MR imaging and the same cellular viability compared with dextran coated SPIOs. The two coating materials coated SPIOs did not affect the cells' activation markers such as cell migration. But the citric-acid coated groups had a higher cytokine production.