Experimental Study Of Dual-targeted Liver Molecular Imaging Using USPIO-loaded Lipid Nanoparticles

Solid lipid nanoparticles (SLN) is a novel nanoparticle delivery system under development in recent years, with solid natural or synthetic lipids such as glyceryl stearate, lecithin, triglycerides as the carrier, the drug or contrast agent encapsulated in the lipid core to form an acid administration system. Because of its strong hydrophobic surface, after intravenous injection into the body, soon to be swallowed by reticuloendothelial system (RES), and into the liver and spleen by passive targeting. Mammalian hepatocytes possess large numbers of high-affinity, cell-surface receptors (asialoglycoprotein receptor, ASGPR) that can bind asialoglycoproteins. It can specifically recognize ligands with terminal galactose residues. Each liver cell has more than5105receptors in normal liver, but the number and function of the receptors was declined in hepatitis, cirrhosis, liver cancer and other liver diseases. Galactose is a hepatocyte-specific ligand of ASGPR and a the liver targeting group, which could induce and improve cell adhesion and the performance of liver extracellular matrix scaffold. In this paper, we prepared lipid magnetic nanoparticles (Gal-SLN-USPIO) with dual-targeting of liver function, which could passive target and active target to live due to active targeting modification in nano-carrier. Then we investigate the targeting effect on normal liver cells and liver cancer, and value the diagnosis of hepatocellular carcinoma, in vivo and in vitro. This study consists of four major sections.Part I:Preparation and physicochemical characteristics of lipid magnetic nanoparticlesThe galactose-stearylamine conjugates (Gal-ODA) was synthesized by chemical coupling reaction between lactose acid (LA) and stearyl amine (ODA) in this research, and proton nuclear magnetic resonance spectroscopy (1H NMR) and Fourier transform infrared spectroscopy (FTIR) was used to confirm its chemical structure. Then SLN-USPIO, Gal-SLN-USPIO/PEG and Gal-SLN-USPIO was prepared, using monoglyceride as lipid materials. Particle size and surface potential of those lipid magnetic nanoparticles were detected by dynamic light scattering using a Zetasizer, and the morphology was examined by tansmission electronic microscopy (TEM). The results show that particle sizes and surface potentials of the3magnetic nanoparticles were about45～60nm and-27～-35mV, and there is no significant differences among them. The three magnetic nanoparticles showed a spherical lipid with uniform size and smooth surface. At room temperature for2weeks the magnetic nanoparticles solutions showed no aggregation. Strengthen external magnetic field adsorption experiments confirmed that the magnetic Fe3O4nanoparticles are wrapped in lipid nanoparticles.Part II:Lipid magnetic nanoparticles in vitro experiments and in vivo studies of liver-targetingFluorescein isothiocyanate (FITC) was used to lable the lipids magnetic nanoparticles in the study; RAW264.7(murine macrophage cell line) cells, LO2(human hepatic cell line) cells and HepG2(human hepatocellular carcinoma cell line) cells were adopted as the active targeting, the passive targeting and the disease models, respectively. Make use of the laser scanning confocal microscopy and the flow cytometer to qualitatively/quantitatively research the uptake of the different magnetic nanoparticles in three cells. Use the four methyl thiazolyl tetrazolium chromatometry method (MTT) to investigate the cytotoxicity of the different lipid magnetic nanoparticles in three kinds of cells. Use a liposoluble DiR as the near-infrared fluorescent dye to prepare the DiR-labeled lipid magnetic nanoparticles and the distribution of the different lipid magnetic nanoparticles in normal mice liver were investigated via the small animals in vivo fluorescence imager. The results showed that the uptake of SLN-USPIO in RAW264.7cells was stronger than Gal-SLN-USPIO and Gal-SLN-USPIO/PEG, and the uptake of Gal-SLN-USPIO in LO2cells was stronger than SLN-USPIO and Gal-SLN-USPIO/PEG, but the uptake of three lipid magnetic nanoparticles in HepG2cells was no significant difference; The LO2and HepG2cells co-incubated competitive uptake experiments showed that the uptake of Gal-SLN-USPIO in LO2cells was significantly stronger than HepG2cells, which had provided the possibility for further in vivo molecular imaging and diagnosis of liver cancer model; When the concentration of three lipid magnetic nanoparticles reached100μg/ml, a variety of cell survival rate were more than80%, which had indicated that the lipid magnetic nanoparticles prepared in this study had a lower toxicity and reliable security in clinical application; The results of in vivo liver distribution indicated that the fluorescence intensity of Gal-SLN-USPIO (with dual-targeting function) in the liver at different time points was stronger than SLN-USPIO and Gal-SLN-USPIO/PEG groups, the results indicated that the dual-targeting Gal-SLN-USPIO was expected to be MRI contrast agent for the diagnosis of the liver disease.Part III:In vitro MRI study of lipid magnetic nanoparticlesMagnetic resonance imaging studies and T2*measurement were carried out on a3.0T MRI scanner. Concentration series from0to100μg/ml of lipid magnetic nanoparticles Gal-SLN-USPIO were prepared in7tubes, respectively. T2*images were acquired with ESWAN sequence, and the results showed that T2*signal intensity decreased significantly with the increase of Fe concentration. T2*time measurement results show that:the T2*time is gradually reduced with the increased concentration of Gal-SLN-USPIO. These results indicated that Gal-SLN-USPIO can be used as contrast agent, and its contrast effect can be enhanced by elevating concentration. LO2cells or HepG2cells were incubated with three different kinds of lipid magnetic nanoparticles for1h, and then scanned with MRI. The results of in vitro MR imaging studies indicated that the intensity of the MR signal was significantly lower after Gal-SLN-USPIO be uptake by LO2cells, and the corresponding R2*value was significantly larger than SLN-USPIO or Gal-SLN-USPIO/PEG group (p<0.05); but there was no significant differences appeared between the three kinds of lipid magnetic nanoparticles after they were uptake by HepG2cells (p>0.05). These results indicated that Gal-SLN-USPIO could be used as a MR probe in in vivo imaging.Part IV:In vivo MRI study of lipid magnetic nanoparticlesIn this study, tumor models were made in orthotopic implantation of HepG2tumor which originated from subcutaneous implantation of HepG2tumor cells. The tumor models were devided into3groups at random:SLN-USPIO group, Gal-SLN-USPIO group and Gal-SLN-USPIO/PEG group. Three different kinds of lipid magnetic nanoparticles (iron dose of about0.5μg/g body weight) were injected into the nude mice via the tail vein. The nude mice were imaged with a small animal coil on a3.0-Tesla MR scanner, and obtained axial T2WI images at different time points. Signal to noise ratio (SNR), contrast to noese ratio (CNR) of tumor and liver were calculated before and after enhancement on all images at different time point. The livers were excised for HE staining to confirming whether the tumor models were success, and Prussian blue staining was used to confirming the targeting distribution of the different lipid magnetic nanoparticles in the liver, respectively. In vivo MR imaging findings indicated that there was no significant difference between three groups before and after the enhanced T2WI images of SNR of the tumor and normal liver tissue (p>0.05); statistical analysis suggested that three groups of normal liver had significant difference in SNR before and after the injection of different lipid magnetic nanoparticles, respectively (p<0.05); The CNR of tumor-liver in Gal-SLN-USPIO group was increased significantly compared with SLN-USPIO group and Gal-SLN-USPIO/PEG group (p<0.05); HE staining results showed that the tumor models of orthotopic implantation of HepG2tumor were constructed successfully; Prussian blue staining showed that, the blue iron particles in Gal-SLN-USPIO group was much more than other two groups, these results were further demonstrated that the ability of targeting liver of Gal-SLN-USPIO was significantly stronger than that of SLN-USPIO and Gal-SLN-USPIO/PEG. The above results suggest that Gal-SLN-USPIO is expected to be a MR probe for liver-specific targeting.Summing up the above experiments, the present study has successfully prepared with active and passive liver targeting function of the lipid magnetic nanoparticles Gal-SLN-USPIO, and in vivo and in vitro experiments confirmed that Gal-SLN-USPIO has a good prospect in the liver targeted molecular imaging in magnetic resonance.