Feasibility Study Of Non-invasively Imaging Early Stage Of Hepatic Fibrosis In Mice By Targeted Multimodal (Magnetic/Optical) Nanoprobe

Liver fibrosis is the common end stage of various kinds of chronic liver damages, which is the public health threaten all over the world. Detection of liver fibrosis in its early stage followed by active interference will control the disease in a certain degree or even invert the disease development. However, there is no effective noninvasive approach to evaluate liver fibrosis in its early stage. Many research works demonstrate that hepatic stellate cells (HSCs) are the primary effector cells to the genesis of liver fibrosis. As HSCs are activated to proliferate, and the neogenesis of intrahepatic neovasculatures takes place, the expression of integrin?v?3 will increase.?v?3 is a kind of extracellular matrix receptor of integrin family. It mediates the adhesion between cell and many kinds of ECMs, having synergistic effect with cytokines. It is one of the major factors in repairing of tissue lesion and matrix remodeling. Subsequence of arginine-glycine-asparagic acid (RGD) is the most effect targeting domain for integrin. And some of the cyclic peptides such as arginine-glycine-aspartic acid-(d-) tyrosine-lysine (RGDyK) and arginine-glycine-aspartic acid-(d-) phenylalanine-lysine (RGDfK) perform even better specific combinative abilities to integrin?v?3 receptor compared to linear peptide. Molecular imaging is a kind of science which describes and measures bioprocesses in cellular and molecular levels in vivo with imaging technologies. Nowadays, molecular imaging is developing fast, each method with its own merits. We benefit a lot to integrate magnetic resonance and optical image systems. The fine spatial resolution of magnetic resonance imaging will make up for the insufficient of optical image. And optical image will also retrieve the limitation of sensitivity to magnetic resonance imaging.Therefore, we conceived to develop a kind of multimodal (magnetic /optical) nanoprobe targeting integrin?v?3 receptor, to dynamically detect the critical events in all the nodes and links along the progress of liver fibrosis development with high resolution and high sensitivity. This essay is divided into five parts according to the design of the project.Part one:Design, synthesis and characterization of targeted multimodal nanoprobeAims:Choose appropriate target, develop multimodal nanoprobes to the diagnosis of mice early liver fibrosis.Methods:Solid-phase synthesis of cRGDyK was followed by purification. Search for the correct route to modify all the peptides, magnetic resonance imaging groups and optical imaging groups to the carrier, which is a kind of dendritic compound called dendrimer, and purify the product. We established a kind of nanoprobe:Den-RGD. We characterized the nanoprobe from all the aspects, including detection of the ratio between modified groups and the carrier by 1H NMR and UV photometer, determine the molecular weight by MALDI-TOF MS and GPC, determine the size distribution and Zeta potential of the nanoprobe, confirm the metallization of the nanoprobe by ICP-AES and their relaxation rates. A kind of control probe, Den-PEG, without targeting peptides, was synsythized and characterized as well.Results:We synthesized both of the nanoprobes successfully. They are positively charged nanoprobes with the size distributions at about 8-12nm and the molecular weights at about 80-95kDa.Conclusions:The synthesis of targeted nanoprobe underlies the foundation for all the experiments in vitro and in vivo.Part two:Evaluation the cytotoxicity and targeting effect of the nanoprobe in cell culture.Aims:Testify the cytotoxicity and targeting effect of the nanoprobe in cell culture.Methods:Improve the enzymolysis and density gradient centrifugation method to separate mice origin primary hepatic stellate cells. Cultivate them and identify the purity with oil red dyeing and desmin and?-SMA immunofluorescence staining. Observe the cytotoxicity of Den-RGD and Den-PEG to mice origin primary hepatic stellate cells through MTT test. Determine the expression of?3 receptor in mice origin primary hepatic stellate cells by western blot technology. Investigate the binding ability of Den-RGD and Den-PEG with hepatic stellate cells using flow cytometry. Observe binding and internalization of Den-RGD and Den-PEG in HSCs under different conditions with confocal microscopy.Results:Modified G5-dendrimers show significantly decreased cytotoxicities to HSCs than before, with the value of IC50 increased from less than 0.5?M to more than 5?M. Activated HSCs express a certain extent of?3 receptors but not so much as U87MG cells do. Den-RGD could quickly bind to mice origin activated primary HSCs, and increased fast as time filed. Its uptake by HSCs was 2.86 times to that of Den-PEG 24h after injection, which could also be competitively inhibited by RGD.Conclusions:All the established nanoprobes are with low cytotoxicity. Nanoprobes can bind to target cells tremendously and swiftly via receptor and ligand effect in vitro.Part three:Establish TAA induced liver fibrosis model in mice and detect the bio-distribution of the target nanoprobes in model mice.Aims:Establish animal models. Precisely determine the time and tissue dependent distribution of nanoprobes in model mice, thus evaluate the feasibility of optical and magnetic resonance images in living animals with these targeted nanoprobes.Methods:We established liver fibrosis model with C57BL/6J mice and created models of three different stages of the disease. H&E staining and Sirius red staining were used for identification. We modified the nanoprobe with 125I, and intravenously inject 5-10?Ci each of the nanoprobes to each mouse (normal and model mice)respectively. We sacrificed the mice 2h and 24h after injection, took out all the major organs of the mouse, like heart, liver, spleen, lung, kidney, appropriate amount of muscle and brain, detected and calculated the unit intensity of radioactivity. We also made target tissues frozen sections to do the autoradiography and quantitative analysis.Results:Nanoprobes mostly load in liver, kidney and spleen.125I-Den-RGD accumulation significantly increases in liver fibrosis model mice liver. Den-RGD aggregation is significantly increased than Den-PEG in model mice livers. The two nanoprobes'unit intensity of radio activities (%ID/g) in liver were 7.96±0.47 vs 5.48±0.33 and 4.22±0.15 vs 1.71±0.13 2h and 24h after injection respectively. Liver tissue section autoradiography outlined trabs shaped signal reinforcement areas in model mice liver, and the quantitative analysis result was identical to the result of bio-distribution.Conclusions:Targeted nanoprobes can accumulate in target tissue with increased quantity and longer time due to the receptor and ligand effect. This will provide the rationale for imaging in living animals.Part four:Trace the distribution of targeted multimodal nanoprobes with optical imaging method.Aims:Take advantage of optical groups in the targeted nanoprobes to trace the distribution of multimodal nanoprobes in living animals and in ex vivo tissues. Trace the relationship between nanoprobes'binding sites and receptors in sections combined with immunofluorescence techniques.Methods:After 24h of intravenous injection of 4.Onmol of Den-RGD and Den-PEG to normal and model C57BL/6J mice, indicate the distribution of the nanoprobes in all the organs with in vivo multispectral imaging system. Observe the sedimentation of the nanoprobes in liver frozen sections under confocal microscopy.Results:The result of mass ex vivo optical imaging is identical to bio-distribution and pathological section results. Den-RGD sedimentation in mice liver increases as the fibrosis degree rise under fluorescence microscope, with a similar pattern to collagen distribution change in liver Sirius red staining. In every stage of liver fibrosis, Den-PEG deposition in liver was dramatically reduced compared to that of Den-RGD, and Den-RGD deposition could be clearly blocked by RGD. Den-RGD shows a good co-localization with?3 receptor antibody and?-SMA antibody in portal areas, fibrous septa and areas around them. Activated IISCs are the main intrahepatic cells which express integrin?v?3 and combine with Den-RGD, and intrahepatic neovessels epithelial cells are taking a small part of the function while macrophages and kupffer cells don't take any part in it at all.Conclusions:NIR influrescence imaging and the results of RGD blockade tests demonstrat that the deposition of the nanoprobe is mainly mediated by the combination between RGD ligands and the integrin?v?3 receptors expressed on the membrane of HSCs, and to some extent by the combination betwwen RGD ligands and the integrin?v?3 receptors expressed on the membrane of intrahepatic neovessels endothelial cells. In addition, immunofluorescence results show that there is a good co-locolization between Den-RGD and either?-SMA or?3, which is the foundation for the following research of MRI.Part five:The primary research in non-invasively evaluating liver fibrosis in model mice with magnetic resonance imaging modal.Aims:Explore the feasibility of non-invasively evaluating liver fibrosis in model mice.Methods:Collect target organs'dynamic T1 weighted image and measure T1 signal intensity before and after intravenously injedted 0.05mmol /kg[Gd3+] of each nanoprobes respectively to normal and model mice on a Bruker Biospec 47/30 small animals magnetic resonance imaging system. The increasement signal in ROI(region of interste) at any time(IE)is expressed by the following equation:IE=(RI(t)-RI(0))/RI(0)×100%, in witch RI (t) represents the standardized signal intensity in given time point. We use the signal intensity of the muscle in the same plane of this time point as a standard, that is RI (t)=T1 (liver) t/T1 (muscle) t. And RI (0) represents the standardized signal intensity before injection, which is standardized by the muscle signal intensity before injuection. Do some stasistical. analysis.Results:There is no difference of liver T1 weighted image signal intensity in normal C57BL/6J mice 24h after intravenously injection between Den-RGD and Den-PEG. There is some increase in liver T1 weighted image signal intensity in 4 weeks liver fibrosis model mice 24h after intravenously injection of Den-RGD compared to Den-PEG (* P<0.05), and the increasement can be blocked by RGD (* P<0.05). There is significant increase in liver T1 weighted image signal intensity in 8 and 12 weeks liver fibrosis model mice 24h after intravenously injection of Den-RGD compared to Den-PEG (** P<0.01), and the increasement can be blocked by RGD (**P<0.01). Group comparisons discover that,24h after injection of Den-RGD, the T1 weighted image signal intensity in 4 weeks model mice liver is higher than that in normal mice (* P<0.05), the T1 weighted image signal intensity in 8 weeks model mice liver is higher than that in 4 weeks model mice (* P<0.05), and the T1 weighted image signal intensity in 12 weeks model mice 1 i ver i s much hi gher than that in 8 weeks model mice (** P<0.01).Conclusions:There is a gradual increasement of T1 weighted signal intensity of MRI due to the targeted nanoprobe from normal mice to early, middle and late stages of model mice. This tendency is obviously related to the digree of the disease. Therefore, this nanoprobe image system cherish the hope as an approach to diagnose liver fibrosis non-invasively with magnetic resonance imaging system in the early stage of the disease.