| Magnetic resonance imaging(MRI)is a very safe imaging technology with good temporal and spatial resolution and without exposing patients to radiation environment Contrast agents usually used in MRI,which function is to disturb the magnetization relaxation process of water molecules around the tested tissue under an external magnetic field,and the transverse relaxation rate or longitudinal relaxation rate of hydrogen nucleus in water molecules,thus improving the signal-to-noise ratio of the acquired image.According to the type of enhancement,contrast agents can be divided into positive contrast agents(T1 contrast agents)and negative contrast agents(T2 contrast agents).T1 contrast agents can brighten the imaging site.Gd(Ⅲ)complexes are widely used on T1 contrast agents,while it shows some disadvantages in vivo.For instance,nonspecific distribution;quickly enters the intercellular space after entering the blood;short half-life;poor cell uptake of diseased cell;high toxicity with free gadolinium ion to biological system,which even induce renal systemic fibrosis.T2 contrast agents are used in MRI to darken the imaging site.These contrast agents are generally ferrite or superparamagnetic iron oxide nanoparticles(SPIONs).Magnetite(Fe3O4)or maghemite(γ-Fe2O3)of inner core and surface modified materials are consisted of SPIONs.Bare SPIONs used in MRI contrast agents have several problems that need to be solved:① poor stability and aggregation;② toxicity of metal or metal oxides on cells;③ SPIONs lead to red blood cell damage after entering the blood,with blood toxicity;④after SPIONs enter the cell,it will cause the increase of intracellular reactive oxygen species(ROS)levels,causing damage to the cell.Surface modifiers not only affect the biocompatibility of nanomaterials,but also plays an important role on the adhesion of biomaterials.The terminal functional group of organic/inorganic surfactants/capping agents are connected to the surface atom of SPIONs through electrostatic interaction or covalent bonds to bio-modify and stabilize the physical properties of exposed SPIONs.Feridex,Conbidex,and Resovist,which were sold worldwide,have been used for liver contrast agents,but they have been withdrawn from the market due to many side effects in clinical use.Among their side effects,the most common is hypersensitivity reaction,which is called complement activation-related pseudo allergy(CARPA).Many studies have reported CARPA possibly caused by iron-based contrast agents surface modified materials,and CARPA has become an indispensable part in contrast agent research due to its universality.The biggest challenge of many iron-containing drugs and contrast agents in clinical application is that they may cause CARPA.Because of the prevalence of CARPA in contrast agents,CARPA has become a problem that needs attention and solution in the development of contrast agentsWidely distributed on the surface of animal cells,inside cells and extracellular matrix,glycosaminoglycans(GAGs),are a class of linear polysaccharides,which are mainly composed of disaccharide repeating units composed of hexosamine(including N-acetylglucosamine and N-acetylgalactosamine)and uronic acid(glucuronic acid or Aidoo uronic acid).GAGs can be divided into glycosaminoglycan and galactoglycosaminoglycan according to the type of hexosamine.The former includes heparin(heparin,Hep)and heparin sulfate(HS),while the latter includes chondroitin sulfate,CS)and hyaluronic acid,HA).GAGs has a wide range of applications in the pharmaceutical field.For example,heparin is the most widely used anticoagulant in the world for the treatment and prevention of thromboembolic diseases.HA can be used as a carrier of hydrogel preparation for regenerative medicine and as a moisturizing ingredient in cosmetics.CS can be used as a therapeutic drug for osteoarthritis and even osteoporosis.GAGs are used as a surface modifier for nanomaterials,and it properties are as follows:high water solubility,no toxicity,biodegradability and no immunogenicity.In addition,GAGs are rich in polyanions,which can stabilize bare SPIONs through electrostatic binding and improve the aggregation characteristics of SPIONs.Iron ions as cations will destroy the phospholipid bilayer of cell membranes,causing hemolysis of red blood cells,while it combination with polyanions can weaken or eliminate the damage or influence of iron ions on cell membranes.Iron-based contrast agents are likely to activate complement through alternative pathways and cause CARPA.Factor H is an inhibitor of alternative pathways,possessing prominent anion binding sites.UFH,CS and HA are polyanions could combine factor H,which can inhibit complement activation.Based on the above,aiming at the problems of poor stability and biocompatibility when SPIONs is used as T2 contrast agent for MRI,the project selected GAGs as modify materials to stabilize SPIONs,including Hep,CS,HA of different molecular weights(including Graded heparin unfractionated heparin,UFH;enoxaparin,Eno in low molecular weight heparin;high molecular weight chondroitin sulfate,HCS;low molecular weight chondroitin sulfate,LCS;high molecular weight hyaluronic acid high molecular weight hyaluronic acid,HHA;low molecular weight hyaluronic acid,LHA The modified SPIONs were screened for MRI imaging in liver cancer model mice,which laid the foundation for the development of effective and safe SPIONs contrast agents.The results and conclusions obtained in this study are mainly as follows1 Preparation and characterization of SPIONs and GAGs-SPIONsSPIONs and GAGs-SPIONs were prepared by co-precipitation.It was systematically characterized,and the specific items are as follows:1.1 Transmission Electron Microscope(TEM)was used to measure their inner core structure,and it was found that SPIONs and GAGs-SPIONs were all black spherical structures under TEM,and SPIONs had obvious aggregation phenomenon On the contrary,GAGs-SPIONs had relatively uniform distribution1.2 Dynamic light scattering method(Dynamic light scattering,DLS)was used to measure the hydrodynamic diameter of GAGs-SPIONs,and it was found that the hydrodynamic diameter of GAGs-SPIONs was about 100nm and the particle size distribution was uniform.Zeta of GAGs-SPIONs The potential value is between-55.4 mV and-23.9 mV,indicating that the colloidal dispersion system has high stability.1.3 Fourier transform infrared spectroscopy(FT-IR)was used to detect the modification of GAGs to SPIONs,and it was found that there are characteristic absorption peaks of corresponding glycosyl functional groups on the spectrum of GAGs-SPIONs,indicating that GAGs were successfully modified on the surface of SPIONs.1.4 X-ray diffraction(XRD)was used to determine the crystal nucleus structure of SPIONs and GAGs-SPIONs.Diffraction peaks of SPIONs and GAGs-SPIONs are both found at 18.44°,30.239°,35.521°,43.340°,53.719°,57.141°,62.879°,74.382°,the positions of these diffraction peaks correspond to the crystalline phases of Fe3O4 in(111),(220),(311),(400),(422),(511),(440),(533),respectively,indicating that SPIONs and GAGs-SPIONs both have trans spinel structure of Fe3O4.1.5 Alternating Gradient Force Magnetic Meter(AGM)was used to measure the magnetic properties of SPIONs and GAGs-SPIONs.It was found that both SPIONs and GAGs-SPIONs had certain magnetic saturation strength,and there was no hysteresis and remanence when the magnetic field was removed,indicating that SPIONs and GAGS-SPIONs were superparamagnetic.1.6 MRI scanner was used to detect relaxation efficiency of GAGs-SPIONs.It was found that the transverse relaxation rates of Eno-SPIONs,UFH-SPIONs,HHA-SPIONs,LHA-SPIONs,LCS-SPIONs,HCS-SPIONs were 85.93 mM-1s-1,192.72 mM-1s-1,368.11 mM-1s-1,424.85 mM-1s-1,852.12 mM-1s-1,949.46 mM-1s-1.It shows that they all have higher transverse relaxation rates than Feridex and Resovist except Eno-SPIONs,indicating the potential to be used as T2 contrast agents.1.7 Preliminary stability expedition.The prepared samples were stored in 4℃ for preliminary stability investigation.It was found that unmodified SPIONs appear precipitation at the second week,but there was almost no change in SPIONs modified by GAGs(GAGs-SPIONs)within three months,indicating that GAGs modification can significantly improve the stability of SPIONs2 Biocompatibility evaluation of GAGs-SPIONs2.1 Cytotoxicity testing of GAGs-SPIONsCCK-8 method was used to detect the effect of GAGs-SPIONs on the survival rate of mouse macrophages.The results showed that UFH-SPIONs,HHA-SPIONs,LHA-SPIONs,LCS-SPIONs and HCS-SPIONs all have the effect of improving the survival rate of mouse macrophages when the concentration of iron ions is 3.12-50μg/mL,and the survival rate of macrophages also increases with the increase of the concentration of iron ions2.2 GAGs-SPIONs uptake by Mouse MacrophagesPrussian blue staining was used to detect the uptake of GAGs-SPIONs by mouse macrophages(iron ion concentration is in the range of 3.12～50μg/mL).The study found that the accumulation of GAGs-SPIONs in macrophages is increased by iron ion concentration.When the iron ion concentration is 25 μg/mL,the number of blue spots in the macrophages of the UFH-SPIONs,LCS-SPIONs,and LHA-SPIONs groups tends to be saturated;when the iron ion concentration is 12.5μg/mL,HCS-SPIONs The number of blue spots in the macrophages of the group of mice tended to be saturated;when the concentration of iron ions was 50μg/mL,the number of blue spots in the macrophages of the HHA-SPIONs group tended to be saturated.This indicates that mouse peritoneal macrophages have good uptake capacity for GAGs-SPIONs,which is the basis for their use as MRI contrast agents2.3 GAGs-SPIONs hemolysis performance testThe hemolysis test found that the hemolysis rate of UFH-SPIONs,LCS-SPIONs and LHA-SPIONs in the range of iron ion concentration of 10～25μg/mL is within the acceptable range possessing good blood compatibility,while HCS-SPIONs and HHA-SPIONs have a hemolysis rate is more than 5%,with poor blood safety.2.4 Complement activation text of GAGs-SPIONs in vitroThe content of human complement complex SC5b-9 and complement factor B Factor(BF)in blood samples after the interaction of LHA-SPIONs,UFH-SPIONs and LCS-SPIONs in GAGs-SPIONs with human serum was detected by ELISA.The study found that,compared with the negative control group,none of the three groups could cause complement activation(P>0.05).2.5 Detection of the effect of GAGs-SPIONs on the release of ROS from macrophagesThe effect of GAGs-SPIONs on the level of reactive oxygen species(ROS)in mouse peritoneal macrophages at an iron concentration of 50μg/mL and 100μg/mL was investigated using an active oxygen detection kit.The study found that at an iron concentration of 50μg/mL GAGs-SPIONs did not cause a significant increase in intracellular ROS levels(P>0.05);and the UFH-SPIONs group also reduced intracellular ROS levels(P<0.01)compared to the negative control group.What’s more,at an iron concentration of 100μg/mL UFH-SPIONs group and LHA-SPIONs group also reduced intracellular ROS levels(P<0.01)compared to the negative control group.2.6 Effect of UFH-SPIONs on mouse coagulation system.The effect of the optimizated contrast agent UFH-SPIONs on the coagulation system of mice was investigated by tail-cutting method in mice.In this experiment,saline as negative control,heparin as positive control,and the human recommended dose of Feridex as reference,each mouse in the experimental group 1 was injected with UFH-SPIONs with a ferric ion mass of 125μg in the tail vein,and each in the experimental group 2 mice were injected with UFH-SPIONs with a mass of 125μg of iron ions into the tail vein of mice.The results showed that the bleeding time of the negative control group was about 300s,and the bleeding time of the positive control group(heparin group)was 1800s.The statistical analysis of the results showed that UFH-SPIONs had no significant effect on the coagulation system of mice(compared to the negative control group P>0.05),indicating that UFH-SPIONs will not cause heparin-induced hemorrhage at the doses.3 Tissue distribution of GAGs-SPIONs in miceSingle tail vein LHA-SPIONs,UFH-SPIONs and LCS-SPIONs(5mg/kg body weight iron ion concentration),the iron content in each tissue sample was detected with the tissue iron determination kit to investigate the GAGs-SPIONs in mice distribution of plasma,heart,liver,spleen,lung,kidney,and brain tissues.The study found that the half-life of UFH-SPIONs,LCS-SPIONs and LHA-SPIONs in mouse plasma were 0.3h,0.24h and 0.46h,respectively,which had the effect of prolonging the plasma half-life of SPIONs compared with literature reported of half-life of SPIONs in plasma;tissues containing macrophages(liver,spleen,Lungs,kidneys,and brain tissue)all have good enrichment capacity for GAGs-SPIONs.The results show that the distribution of GAGs-SPIONs in above mentioned tissues is helpful for MRI imaging enhancement.4 MRI imaging of GAGS-SPIONS in mouse liver cancer modelReplicate the liver cancer model of nude mice and administer GAGs-SPIONs(including LCS-SPIONs,UFH-SPIONs,LHA-SPIONs)with a concentration of 5mg/kg ferric ion to each mouse by tail vein administration.T2 weighted MRI images before and 15,30,60,90,120min after administration were collected using magnetic resonance scanner.The signal-to-noise ratio parameters of each image are collected,and the image of signal-to-noise ratio changing with time after drug administration is drawn.The research results show that the contrast effect of nude mice with liver cancer model is significant within 1 hour after administration,and the best contrast effect is achieved around 60min.The signal-to-noise ratio of MRI images showed a trend of rising first and then falling.At 60 minutes after administration,the signal-to-noise change ratio of LCS-SPIONs,UFH-SPIONs and LHA-SPIONs images of nude mice all reached the maximum,increasing by 50.68%,67.22%and 71.75%respectively compared with that before administration.The results show that GAGs-SPIONs(LCS-SPIONs,UFH-SPIONs,LHA-SPIONs)all have a good ability to improve the contrast of MRI imagesIn conclusion,the modification of SPIONs by GAGs in this subject can effectively improve the problems of poor stability,poor biocompatibility(cytotoxicity,hemolysis,CARPA reaction,ROS release)and short half-life in vivo in the application of bare SPIONs.The GAGs-SPIONs prepared in this project retain the superparamagnetic properties of SPIONs.After in vivo distribution and evaluation of tumor-bearing mice MRI imaging effects,LHA-SPIONs,UFH-SPIONs and LCS-SPIONs are considered to be effective and safe MRI T2 imaging,and they are expected to be used in MRI detection of liver and other macrophage-rich organs. |
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