Experimental Study Of SiO₂@Fe₃O₄ Nanoparticles Labeled Rat Bone Marrow Mesenchymal Stem Cells In Vitro

Background:With the progress in regenerative medicine and tissue engineering, stem cell therapy has become a new and attractive therapeutic approach for treatment of diseases. Bone marrow mesenchymal stem cells（BMSCs） were regarded as a promising cell for stem cell therapy because of their special superiority, including easy separation and culture, low immunogenicity, avoidance of ethical problems, ability to promote the advantages of cellular implantation and homing properties, etc. Some studies have found that transplantation of autologous or allogeneic BMSCs in both animal experiments and clinical trials there would be no serious adverse reactions, transplant-related tumor formation and mortality. The biological safety of BMSCs transplantation has been testified by domestic and international scholars.However, a serious problem has become a major obstacle to the development of BMSCs transplantation. That is we couldn’t get accurate and continuous information of the transplanted BMSCs in vivo because of the difficulty of dynamically tracking the cells in long-term. In recent years, a large number of researchers have proved that BMSCs are made MR-visible by labeling with superparamagnetic iron oxide（SPIO） particles, and this method is expected to solve the above problem effectively.Fe₃O₄@SiO₂ nanoparticles have superparamagnetism and can be used as a negative MRI contrast agent. Moreover, the silicon dioxide layer of Fe₃O₄@SiO2 nanoparticles can increase the particle size and dispersion, improve histocompatibility, reduce toxicity, and prevent them from being broken down into iron ions to lose superparamagnetic. So this specific structure ensures Fe₃O₄@SiO2 nanoparticles can label cells for longer time. In addition, we can make the shell into mesoporous silica which can load small molecules like drugs by controlling the synthesis condition. Therefore, using the BMSCs labeled with mesoporous silica magnetic nanoparticles（M-MSNs） carrying a particular drug not only can we dynamically monitor the biological behavior of BMSCs in vivo. We can also realize the goal of targeting drug delivery through magnetic guidance and homing properties of BMSCs. Finally, we can achieve a better therapeutic effect by using the combination treatment of stem cell transplantation and drug therapy. Objective:In this study, rat BMSCs and Fe₃O₄@SiO2 nanoparticles were took as experimental materials to explore the feasibility and optimal marking method of labeling BMSCs with Fe₃O₄@SiO2 nanoparticles. According to the results of MRI, the optimum number of cells that can be imaged clearly on MRI would preliminary to be determined. In addition, the experimental evidence would laid basis for cell tracing in vivo and clinical application of BMSCs transplantation. Methods:1. Primary rat BMSCs were isolated by the blood marrow adherent culture method. Then we got sufficient and pure cells after several subcultures.2. To identify the rat BMSCs we obtained, cell morphology was observed by inverted microscope, surface antigens were tested by flow cytometry, and pluripotency was detected by adipogenic and osteogenic differentiation.3. Fe₃O₄ nanoparticles were synthesized by chemical co-precipitation method. Then the nanoparticles coated with a layer of SiO2 by Stober method to form Fe₃O₄@SiO₂ nanoparticles. Examine their morphological characteristics through the transmission electron microscope.4. The marked media with different iron concentration（0, 25, 50, 100, 200 μg/ml） were prepared to incubated with BMSCs for 24 h, then observed the cells and calculated the labeling rate of Fe₃O₄@SiO2 nanoparticles after prussian blue staining.5. Cell viability of rat BMSCs incubated with the different marked media was observed by trypan-blue exclusion at different time points.6. Cytotoxicity of Fe₃O₄@SiO₂ nanoparticles was detected by CCK-8 test and assessed the effect of them on cell proliferation.7. At the optimum labeling concentration and time, different numbers of Fe₃O₄@SiO₂ nanoparticles-labeled rat BMSCs were imaged by MRI. Results:1. Rat BMSCs were isolated successfully from bone marrow adherent culture and obtained uniform morphology cells when cultured to the third generation. And enough cells achieved for the following experiments.2. The cells adhered to the bottom of the dish were spindle-shaped, and their growth characteristics were in accord with BMSCs features. The flow cytometry results showed that the cells were high expression of MSCs surface markers CD44, CD90, and low expression of CD34, CD45. The adipogenic and osteogenic differentiation experiments suggested that the cells can be successfully differentiated into the adipogenic and osteogenic cells.3. The prepared Fe₃O₄@SiO2 nanoparticles were mainly spherical and the particle size is about 50¹00 nm which have superparamagnetic and good dispersion in water. The cores of them consist of single or multiple Fe₃O₄ nanoparticles and the outer layer was SiO2.4. Prussian blue staining showed Fe₃O₄@SiO₂ nanoparticles can be uptaken by BMSCs and concentrated in the peripheral edge of the nuclear membrane; with the increase of the iron concentration the blue dye particles in the cytoplasm went darker; the labeling rate and the iron concentration positively correlated each other, the labeling rate of different groups are all more than 95%, and compared with 25 μg/ml the others group has significant statistically difference.5. Trypan blue exclusion demonstrated that cell viability obviously reduced only in 200 μg/ml iron concentration group at 24 h, and has significant statistically difference compared with the control group. CCK-8 test results showed that Fe₃O₄@SiO₂ nanoparticles began be toxic on rat BMSCs after being labeled 24 h on the iron concentration of incubation reached 100 μg/ml.6. The minimum cell concentration of BMSCs（incubated with of 50 μg/ml iron concentration for 24 h） that could be detected by 1.5T MRI with T2 scanning sequence was 2.5×105/ml, and the signal reduced more obvious with the increase of labeled cell concentration. Conclusions:1. Uniform, stable and superparamagnetic Fe₃O₄@SiO2 nanoparticles can be synthesized through Chemical co-precipitation method and Stober method.2. Rat BMSCs can be successfully labeled with Fe₃O₄@SiO₂ nanoparticles, and in a certain concentration（25 50 μg/ml）, Fe₃O₄@SiO2 nanoparticles was non-toxic and have no side effects on rat BMSCs.3. Rat BMSCs labeled with Fe₃O₄@SiO2 nanoparticles can be clearly imaging in MRI with T2 scanning sequence, which laid experimental basis for the further BMSCs transplantation tracing research in vivo.