Construction Of A New Type Of SPION Especially For MSCs Labeling And In Vivo Tracking With MRI

Background and Objective:The limitations of therapeutic regimes and efficacy in the management of malignant tumors have always been a great challenge in the field of clinical oncology. The essential problem lies in the lack of specificity in traditional therapy and the restriction of medication dosage due to systemic toxicity in chemotherapy. Researchers have been working on developing new therapeutic strategies and methods, hoping to conquer the barriers. Recent researches have suggested that mesenchymal stem cells (MSCs) have inherent tropism for malignant tumor cells and low immunogenicity. Therefore, MSCs could be used as a "Troy" to deliver genetic agents or drugs directly to the tumor tissues, and they also could be labeled with contrast agents for cell tracking and real-time monitoring of tumors. A lot of obstacles will be tackled with the targeted-delivery vehicles, MSCs, in the diagnosis and treatment of malignant tumors, which concerning safety, immunogenicity of vehicles and transgenes, and systemic toxicity. Excellent in vivo cell tracking technologies have to be adopted to assist in vivo application of cellular vehicles. Magnetic resonance imaging (MRI) can provide good spatial resolution and great contrast between different soft tissues. It consists of multiple sequences and will provide images from multiple angles. Therefore, MRI has become one of the most popular non-invasive examination techniques in clinical practice. Novel MR contrast-agents, especially superparamagnetic iron oxide nanoparticle (SPION) technique have experienced rapid development in recent years. SPION technique has become an optimal choice for performing in vitro labeling of MSCs and in vivo cell tracking in tumor treatment. Iron is a magnetically susceptible material. When SPION is used as a negative contrast media in labeling of implanted cells (either intracellular labeling or cell surface labeling), it will significantly lower the MR signal intensity in T2-weight imaging (T2WI) and T2*-weighted imaging (T2*WI), thus the implanted cells and original tissue cells can be distinguished from each other easily. At last, the movement and distribution of the SPION-loaded cells can be identified indirectly. Two particular SPIONs, Ferumoxides (Endorem(?) in Europe and Feridex(?) in the USA and Japan) and Ferucarbotran (Resovist(?) in Europe and Japan) have been approved by FDA as clinical grade contrast agents for enhancing MRI detection. However, they are only reticuloendothelial system-specific contrast agents. The MR imaging will be monitored on a short-term basis because Kupffer cells (specialized macrophages located in the liver) have a powerful function of uptake of foreign particles and the coated dextran or its derivative is easily to be metabolized and excreted. As to labeling of MSCs with SPION, the uptake of general SPION by cells is very low, which results in insufficient iron load in single cells, and the quick cell division will lead to rapid dilution of iron load in single cells. Therefore, it is obvious that the current SPION products can't meet the requirements for contrast media in long-term dynamic MR monitoring.In consideration of the urgent need for molecular labeling of MSCs and on the basis of the worldwide research outcomes and latest development in labeling of cells with SPION, the objective of this study was to design and synthesize a new type of SPION (SPIO@SiO2-NH2), which is specialized in labeling nonphagocytic cells with great proliferative capacity such as MSCs for MR tracking.The design philosophy includes:1) Fe3O4 crystal is used as the core of SPIO@SiO2-NH2, which makes the SPION a superparamagnetic substance.2) The coating material SiO2 contributes to the good hydrophilicity, stability, water dispersity and biocompatibility of the SPION. In addition, the chemical inertia of SiO2 can protect SPIO@SiO2-NH2 from degradation by the enzyme or acid in lysosomes, which allowing a long-term existence of the SPION in cells.3) NH2 groups will be attached to the surface of SiO2 material, and the positive charges they are carrying can be acted with the negative charges on the surface of cells due to the forces of electric attraction. This will increase the cellular uptake of SPIO@SiO2-NH2, leading to increased iron load in cells which is a guarantee of satisfactory MR imaging.4) 3-aminopropyltriethoxysilane (APTES) is directly employed to modify the surface of Fe3O4 crystal core of the SPION. The thin coating of SiO2 will have very little effect on saturation magnetic induction of the particles.Experimental summary and methods:1) The Fe3O4 nanoparticles (core of the SPION) with good dispersity were produced via coprecipitation method. APTES was directly used to modify the surface of SPIO nanoparticles, resulted in a SPION with an amino functional group (SPIO@SiO2-NH2).2).Transmission electron microscope (TEM), X-ray diffractometer (XRD), magnetic induction detection, and relaxation rate measurement were adopted to identify the physical and chemical properties of the newly synthesized nanoparticles.3) The MSCs were isolated from the bone marrow of rabbits with adhesion method. The in vitro cell labeling was performed with the novel SPIO@SiO2-NH2.4) Prussian blue staining, inductively coupled plasma optical emission spectrometer (ICP-OES) and MRI scan were adopted for confirming the effect of in vitro SPIO@SiO2-NH2 labeled MSCs (short-term and long-term). Trypan blue staining was adopted for detecting the impact of the nanoparticles on the activity of MSCs. Methyl thiazolyl tetrazolium (MTT) method was applied to detect the impact of the nanoparticles on the proliferation of MSCs and also the impact on the differentiation potential of MSCs.5) Rabbit models of brain damage and erector spinae muscle damage were established. The SPIO@SiO2-NH2 labeled MSCs were locally injected into the rabbits and MRI was used for long-term dynamic monitoring of the MSCs. After that, the tissue slices were co-stained with Hematoxylin and eosin (HE) and Prussian blue to detect the outcomes of cells and nanoparticles.Results:1) A new type of SPION (SPIO@SiO2-NH2) was successfully synthesized, and at the same time SPIO@SiO2 was synthesized as a control.2) SPIO@SiO2-NH2 possessed a polyhedral shape with a diameter of 6-11 nm. The magnetic induction intensity and relaxation rate were 52.5 emug-1 Fe and 43.5±9.1 mm-1 s-1, respectively.3) Rabbit bone marrow-derived MSCs were successfully isolated. The incubation of MSCs with SPIO@SiO2-NH2 of 4.5μg[Fe]/mL for 16 hours showed a good labeling outcome (no necessary for adding transfection reagent).4) SPIO@SiO2-NH2 with the concentration of 4.5μg[Fe]/mL could be used for high-quality MSC labeling. The MSCs were located in the lysosomes or vesicles in cytoplasm after the uptake by cells. The iron load was 68.7±11.2 pg/cell (17.4±3.9 pg/cell of SPIO@SiO2 would be needed for equal iron concentration). The threshold concentration of MSC for 3.0T MRI was 1,000 MSCs (5,000 cells would be needed for equal iron concentration). There was no significant impact on the activity, proliferation or three lines of differentiation potentials (osteogenic, adipogenic and chondrogenic differentiations) of MSCs after labeling with SPIO@SiO2-NH2. No apoptosis or necrosis outcome had been found during the experiment. At day 33 (natural cell passage) after labeling with SPIO@SiO2-NH2, a small amount of nanoparticles still could be detected in the lysosomes or vesicles. The threshold concentration of MSCs for 3.0T MRI was 10,000 cells (50,000 SPIO@SiO2-labeled cells would be needed for equal iron concentration). 5) The rabbit models of brain damage and erector spinae muscle damage were successfully established. The SPIO@SiO2-NH2 labeled MSCs were locally injected into the rabbits. The tissues were well-displayed by MR imaging and the long-term (8-12 weeks) dynamic monitoring of MSCs was accomplished. The detection of tissue slices co-stained with HE and Prussian blue demonstrated that the blue-stained nanoparticles were located in the injected cells. However, whether the cells had experienced differentiation (and if yes, what types of the differentiated cells) was still not clear.Conclusion:The new type of SPION (SPIO@SiO2-NH2) which was constructed according to our design has good physical and chemical properties; it can effectively label the MSCs derived from the bone marrow of rabbits in vitro with low iron concentration, and at the same time it doesn't affect the viability, proliferation, apoptosis or necrosis status and differentiation potential of MSCs. Amine modified SPION could improve the ability of uptake of MSCs. Locally injecting of SPIO@SiO2-NH2 labeled MSCs contributes to long-term dynamic monitor of tissues with MRI.