Experimental Study On Relative Magnetic Properties And Molecular Imaging Of HSA-Dopamine-Fe3O4 Nanoparticles

Background and purposeMolecular imaging is a rapidly growing biomedical research tool and clinical discipline aimed at noninvasive,quantitative visualization of in vivo molecular processes occurring at cellular and subcellular levels.In general, molecular imaging applications require fast, sensitive and high-resolution imaging techniques, combined with the use of contrast agents that enable the visualization of biological processes at the cellular and molecular level.Until recently, positron emission tomography (PET), single photon emission computed tomography (SPECT) and optical imaging techniques were the most frequently used imaging modalities for pre-clinical molecular imaging in vivo. This is predominantly attributed to the high sensitivity of these modalities,However, nuclear imaging techniques (PET/SPECT) use ionizing radiation, provide no anatomical information and have a low spatial resolution. And Optical imaging suffers from limited depth penetration and relatively low spatial resolution.Therefore,magnetic resonance imaging (MRI) has been recently introduced as an alternative, as this technique provides excellent intrinsic soft-tissue contrast at a high spatial resolution without using ionizing radiation. Moreover, this technology has the advantage that both anatomical information and physiological parameters, like diffusion, perfusion and flow, can be obtained within one single experiment by combining different imaging sequences.MR has become an important tool for molecular imaging.The drawback of MRI is however its low sensitivity for MR contrast agents. Nevertheless, different strategies can be applied to improve the efficiency of such agents, and thus make MRI suitable for molecular imaging purposes. The application of magnetic resonance imaging in conjunction with iron oxide nanoparticle (IONP) based cell labeling techniques, has provided an excellent solutionto the non-invasive tracking of the implanted cells in the host organism. One major challenge for this technique, however, is to induce sufficient amount of particles into the cells to compensate the dilution effect caused by cell division, while not affecting the normal cellular functions. For cell labeling, although SPIO particles are easily internalized by macrophages, uptake by nqnphagocytic and slow-dividing cells is poor, so that high concentrations which often cause cell toxicity are needed for efficient cell labeling.Currently, Several approaches have been raised and investigated to improve the internalization of the contrast agent, with the mostly utilized one being the employment of commercial Feridex in combination with transfection agents. However, lacking of a universal formula, tedious trials and errors have to be made with individual cell line to achieve optimal transfection.In view of these factors, In the study, we conducted the study of molecular imaging with MR imaging and a novel magnetic resonance contrast agent with high magnetic moment-HSA-Dopamine-Fe3O4 nanoparticles (HSA-IONPs) for. through comparative study of Feridex and HSA-Dopamine-Fe3O4 nanoparticles, we investigaed magnetic properties of HSA-IONPs and cell labeling efficiency for different types of cell lines (final concentration of 20μg·ml-1 Fe), and performed molecular magnetic resonance Imaging using neural stem cells,macrophages labled with HSA-IONPs in different animal models.Purpose To synthesize HSA-Dopamine-Fe3O4 nanoparticles (HSA-IONPs)-a novel magnetic resonance contrast agent for molecular imaging and study its magnetic properties.Methods Olecate coated iron oxide nanoparticles (15 nm) were obtained from Ocean NanoTech (Springdale, AR). For surface modification, about 50 mg of oleate coated nanoparticles were dispersed in 5 ml of chloroform. Into the solution,20 mg of dopamine in 2 ml DMSO was added, forming a homogeneous solution. The mixture was heated at 70℃for 1 hour, and was then cooled down to r.t. Hexanes was added as poor solvent to precipitate the nanoparticles, and the nanoparticles were collected by centrifuging at 15,000 g for 15 minutes. Afterwards, they were blow dried with nitrogen, and were redispersed in DMSO with the aid of sonication. On the other hand,20 mg of HSA was dissolved in borate buffer (50 nM, pH 8.5). And with sonication, the nanoparticles in DMSO were dropwise added to the HSA solution. get rid of free dopamine and free HSA.then,were redipersed in PBS buffer. Nanoparticle were quantified and characterized by ICP-AMS,TEM,DSL(dynamic light scattering) and MRI. After normal nude mice were injected with Feridex and HSA-IONPs (10mg Fe/kg) via tail vein.T2W MR scan were performed for studying liver imaging in vivo at different time points.Results Dynamic light scattering (DLS) measured the hydrodynamic diameter of the HSA-IONPs in their dispersion state, The measurement results in water were 28.4 nm. TEM images showed Nanoparticles were monodisperse and single crystalline with circular structure, no aggregated clumps. The results of zeta potential measurements revealed that the HSA-Dopamine-Fe3O4 nanoparticles exhibited negative zeta potential (-9.46±1.86MV).MR phantom results indicated that R2 value of HSA-IONPs(R2=314.5mM-1S-1) was significent higher than that of the conventiona-lly utilized Feridex nanoparticles (R2=123.6mM-1S-1), those HSA-Dopamine-Fe3O4 show better T2 contrast and can be used for liver imaging.Conclusion HSA-IONPs have higher magnetic moment and are better than commercial SPIO nanoparticles(Feridex).Purpose To study cell labeling efficiency of HSA-IONPs as a novel magnetic resonance contrast agent for molecular imaging. Methods HSAIO-NPs or Feridex(final concentration of 20μg·ml-1 Fe) was used to label human prostate cancer PC-3 cells,human head and neck squamous cell carcinoma UMSCC-22B cells,neural stem cells,mesenchymal stem cells,human embryonic stem cells and macrophages respectively at different time points (2,9,12,24h or 48h), All Cells were stained by prussian blue and then counterstained with nuclear fast red. MTT assay was used to evaluate the in vitro cytotoxic effects of nanoparticles. Both nanoparticles phagocytosed by neural stem cells and macrophages were quantified and characterized by ICP-AMS,TEM,and MR Phantom imaging.T2W MR scans were performed 6h after labeled neural stem cells were implanted into bilateral basal ganglia of nude mouse brains by stereotactic injection.24h after the onset of MCAO, macrophages labeled by HSA-IONPs were injected into rats via tail vein and MR scan were performed at different time points (prescan,4h, 1d,2d,5d,7d).Results All cells were incubated with merely 20μgml particles without transfection agent for efficient cell labeling.TEM analysis results found populations of nanoparticles within endosomes/lysosoms and some aggregation could be seen. no toxicity and no harm on cell growth and division was found for the studied cell lines (P<0.05).The results of ICP-AMS assay showed that neural stem cells and macrophages labeled with HSA-IONPs presented higher uptake of nanoparticles than those labled with Feridex. Neural stem cells were incubated with HSA-IONPs or Feridex plus transfection reagents(50μg Fe·ml-1 plus Lipofactamine 2000) for 24h. Compared with Feridex nanoparticles, neural stem cells labeled with HSA-IONPs and transfection agents presented higher uptake of nanoparticles. There was strong dependence on the concentration of nano-particles and iron uptake of neural stem cells. MR Phantom images showed a significant difference between T2WI signal intensity of cell labled with HSA-IONPs and feridex. There was strong linear correlation between R2 values and number of cells before the saturation limit.In vivo MR imaging and pathological examinations confirmed neural stem cells incubated with HSA-IONPs absored more iron than those incubated with Feridex. MR scans could be used to track macrophage migration after focal cerebral ischemic stroke.Conclusion Cell labeling efficiency of HSA-IONPs has an advantage over those of Feridex nanoparticle.Purpose To explore the feasibility of MR imaging for tumor-associated macrophages.Methods Macrophages(RAW264.7)were incubated with HSA-IONPs (20μg/ml Fe)for 24 hours,Then,5×106 labeled macrophages were injected into subcutaneous 4T1 tumor-bearing BALB/c mice and 22B tumor-bearing nude mice via tail vein. At different time points(0h,6h,1d,2d,3d or 7d), all animals performed transverse and coronal T2-weighted MR scans using a 7.0-T MR imaging unit..After sacrifice at different time points, Tumor,liver,spleen and other organs were removed and processed for histological examination.Results Prussian blue staining,TEM and cell phantom imaging confirm efficient cell labeling. in 4T1 tumor model,MR images showed significant low signal intensity on T2-weighted imaging surrounding the center of necrosis and cystic degeneration 6 hours after macrophage injection.,irregular ring low signal enhancement could be seen at the junction of necrotic and parenchyma area at 24 hours. In 22B tumor model,MR images presented distinct multiple small scattered foci of low signal intensity on T2-weighted imaging around the center of necrosis and cystic degeneration 6 hours after macrophage injection. at 24 hours, reduced multiple small scattered foci of low signal intensity could be observed.2,3 or 7days after macrophage injection, MR morphology and location of low signal intensity was similar to those at 24 hours after injection on T2-weighted imaging.But, signal intensity gradually dereased over time.pathological examination verified the presence of labeled macrophages, pathological findings were consistent with imaging results.Conclusion MR imaging for tumor-associated macrophages is feasible.24 hours after macrophage injection is the optimal time point for studing macrophage migrating and infiltrating into tumor on T2-weighted imaging. MR images may indicate the perfusion of local blood flow and the status of neovascularization 6 hours after macrophage injection, The pattern of TAM distribution differed between different tumors.