Long-term And Biodegradable Manganese Oxide-caramelization Nanosphere:MRI Enhancement Of Hepatobiliary Tumor And The Mechanism

BackgroundPrimary liver cancer,a malignant tumor origin in the hepatic cells or intra-hepatic bile duct epithelial cells,is one of common malignant tumor in China.Epidemiological studies have shown that HCC ranks third tumor in the whole world,and prevalence of liver cancer ranks the first as well as the-second cancer lead death in China.Because surgical resection is the best therapeutic treatment for the early HCC nowadays,early diagnosis play important role in the HCC.Imaging diagnosis includes ultrasonography(US),Computed Tomography(CT),Magnetic Resonance Imaging(MRI)and Positron Emission Tomography(PET).MRI,one of the noninvasive diagnostic modalities,can acquire three-dimensional tomographic information for whole tissue samples with high intensity resolution,which has attracted much attention in the diagnosis of liver cancer.Contrast agents can enhance the tissue contrast to increase diagnostic accuracy.There are two forms of contrast agents used in MRI:paramagnetic contrast agent and superparamagnetic contrast agent.Gadolinium and manganese based agents,the representative of the paramagnetic contrast agents,is mainly used for T1WI imaging,for example,Megnevist was clinically used.Iron-based agent,the representative of the superparamagnetic contrast agent,is mainly used for T2WI imaging,such as SPIO.Application of gadolinium-based contrast agents can lead to renal fibrosis,while SPIO can reduce the T2WI intensity,which may lose diagnosis information.Thus,many domestic and foreign researchers pay attention to the manganese-based contrast agent.Although manganese has toxicity in neurological and circulatory system,some studies have shown that low doses of manganese oxide are not cause Parkinson's disease and cardiac toxicity.In addition,the manganese ions has more unpaired electrons outside the nuclear,so it has stronger T1-weighted effect than gadolinium ions.Dextran is a hydrophilic polysaccharide composed of glucose,which has good biocompatibility.It can increase the longevity of therapeuticagents in the circulationas a macromolecular carriers uesd as drug and protein transportors.In addition,dextran is mainly degraded in the cellular lysosome and a little amount of it is degraded by glucosidase as well as excreted by bile duct.Studies have found that the more increase the Mw,the faster dextrans metabolism.Based on thesis above,we have prepared the manganese oxide-caramelizati on nanosphere(Mn@CNS)for MRI-weighted imaging of hepatobiliary malignanc ies.It has the following features:first,CNS can increase the longevity of manga nese oxide in the circulationas a carriers to achieve MRI long-term delayed enhancement.Two,Mn@CNS can be degraded by intracellular lysosomes,which has potential pH-response to achieve properties of tumor-targeting and bio-degradiable.Three,because of EPR effect,long-term circulation and pH-response,nanos pheres can avoid the phagocytosis of reticuloendothelial cells in the liver and spleen and realize MR T1-weighted imaging in hepatobiliary malignancy.AimTo examine the possibility of developing a long-term and biodegradable MRI contrast agent-manganese oxide-caramel ization nanosphere(called as Mn@CNS)used for diagnosis of hepatobiliary carcinoma and the imaging mechanism.Method1.Construction and characterization of nanospheres:CNS displayed nanospheres made up of glucose monomers by a hydrothermalmethod.Manganese oxide combined to CNS via chemical bond forming Mn@CNS.The size of the nanospheres was observed by transmission electron microscopy(TEM),and the surface functional groups were analyzed by infrared spectroscopy.The relaxation rates r1,and r2 of the nanospheres were measured by MR T,mapping and T2 mapping compared with that of Gd-DTPA Relaxation rate.2.In Vitro experiments:The viability of Mn@CNS in HepG2 lines was tested by CCK8 cytotoxicity test.The uptake by the macrophage cell line RAW264.7,the rat liver cell line BRL-3 A and the human hepatocellular carcinoma cell line HepG2 were measured by inductively coupled plasma mass spectrometry(ICP-MS).Flow cytometry was performed to determine whether sodium dextran sulfate inhibits competitively the fluorescein isothiocyanate(FITC)labeling Mn@CNS nanosphere.The expression of scavenger receptor(ScR)in RAW264.7,BRL-3A,and HepG2 after the Mn@CNS injection was determined by Western Blot.TEM observed the interlization of Mn@CNS in RAW264.7,BRL-3 A and HepG2.The localization of FITC-Mn@CNS in RAW264.7,BRL-3A and HepG2 was observed by laser scanning confocal microscopy.3.Animal experiments:SD rats were induced into Hepatocellular Carcinoma and Intrahepatic Cholangiocarcinoma by DEN and TAA.Rats bearing the HCC were used to observe enhancement effect in different time points after Mn@CNS injection via caudal vein.The repeatability of Mn@CNS enhancement effect enhancement was further confirmed in intrahepatic cholangiocarcinoma group.Compared with Mn@CNS,delayed MR enhancement was observed in rats bearing raintrahepatic cholangiocarcinoma after Gd-EOB-DTPA injection via tail vein.After the MRI scanning,the rats were sacrificed and the liver was dissected.TEM was used to observe the excretion pathway of Mn@CNS in rats bearing hepatobiliary malignancy at different time points.Western Blot was uesd to measure tha change of divalent metal ion receptor(DMT-1)and transferrin receptor(TfR)in Balb/c mice liver after Mn@CNS injection.To study the metabolism of the dextran,the PAS staining were implemented in the liver specimens from the rats bearing HCC.Results1.The TEM results showed that the diameter of CNS and Mn@CNS were 140 nm and 160 nm.The figure showed that the nanoparticles surface was coated with black ring,which indicating that manganese oxide have successfully coupled with caramelized dextran.The infrared spectrum showed that the peak of the Mn@CNS was at 3300 cm-1,which contribute to the hydroxyl groups on the Mn@CNS surface.To quantitatively evaluate the MR contrast enhancements,thelongitudinal(r1)and transverse(r2)relaxivity values of Mn@CNS and Gd-DTPA were compared and calculated through the curve fitting of relaxation time versus the Mn and Gd concentration.T,mapping and T2 mapping showed that r1 and r2 of Mn@CNS nanosphere were 11.63 and 41.36 mM-1s-1,respectively,as well as r1 and r2 of Gd-DTPA were 4.11 and 4.82mM-1s-1,respectively,which indicating that the Mn @CNS nanosphere has a higher relaxation rate than the Gd-DTPA.2.Cellular viability test CCK8 has shown that HepG2 was incubated with Mn@CNS at different concentrations(25,50,100,200 ppm)for 12 h,24h,36 h and 48 h,the cellular viability ranged from 80%to 120%,indicating that Mn@CNS has no obvious cytotoxicity.3.To compare the cellular pre-and pro-intake of Mn@CNS among RAW264.7,BRL-3A and HepG2,manganese content were measured by ICP-MS.The pre-intake and pro-intake content of manganese of RAW264.7,BRL-3A and HepG2 are 18.69±1.27 and 21.51±0.8,20.03±1.13 and 38.17±1.93,11.71±1.22 and 63.67±3.54 pg percell,respectively.4.The results of competitive inhibition experiments show that as the amount of sodium dextran sulfate increase from 1 to 50 times RAW264.7 significantly reduces the intake of FITC-Mn@CNS,which indicating a competitive inhibition of ScR by sodium dextran sulfate.5.Western Blot results showed that Mn@CNS significantly up-regulated the expression of ScR in RAW264.7(p<0.05),while it had no effect on the expression of ScR in BRL-3A and HepG2,which indicating that nanoparticle recognized by RAW264.7 was dependent on ScR but BRL-3A and HepG2 may recognize nanoparticle in other ways.6.Cellular TEM observed the uptake of Mn@CNS by RAW264.7,BRL-3A and HepG2 at different time,then forming vesicles also called endosomes and finally fusion with lysosomes,indicating that the nanoparticle could be uptook by the three cell lines and degraded by lysosome.7.Laser confocal microscopy also revealed that FITC-Mn@CNS located in cellular lysosomes of RAW264.7,BRL-3A and HepG2.8.The results of vivo MRI showed that the delayed enhancement in rats bearing HCC could sustain for 1 day,and the enhancement peak appeared at 3 hours after nanoparticle injection.Whereas,the delayed enhancement in rats bearing intrahepatic cholangiocarcinoma could susutain for 2 days and the peak also appeared at 4 hours after injection.There were no significantly delayed enhancement in rats bearing intrahepatic cholangiocarcinoma at two hour after Gd-EOB-DTPA injection.9.PAS staining results showed that intrahepatic caramelized dextran in the HCC was high within 3 hours after injection and returned to the initial state after one day,indicating that caramelized dextran can be degraded by lysosome.10.TEM of hepatocarcinoma and intrahepatic cholangiocarcinoma showed that Mn@CNS was uptook into hepatocarcinoma cells and there were no Mn@CNS present in small bile ducts,suggesting that Mn@CNS was mainly metabolized by lysosomal degradation.11.Western blot analysis of TfR and DMT-1 showed that DMT-1 in Balb/c mice was significantly up-regulated(p=0.002),while no significant up-regulation of TfR was observed,indicating that the metabolism of intrahepatic manganese depended mainly on DMT 1.ConclusionMn@CNS was successfully built by the hydrothermal method,which had characters of high T1 relaxation,low cellular toxicity,bio-degradable and long-term delay enhancement.Nanoparticles were mainly taken up by liver cancer cells and macrophages as well as degraded in lysosomes,which showed nanosphere could avoid macrophage uptake and accumulate in tumor cells.MRI T1-weighte d showed that the tumor enhanced distinctly after the Mn@CNS injection whose delayed enhancement time was higher than the Ultravist-hepatobiliary specific contrast agent.