Synthesis Of Protein-based MR Nanoprobe With High Biocompatibility And Its Molecular Imaging Study

Objective: This study was aimed to prepare a protein-based gadolinium oxide nanoparticle by simulating biomineralization with good magnetic resonance imaging performance?low toxicity to organisms a high degree of biocompatibility and clearance.This study was intended to use clinically-enhanced MR angiography to evaluate the blood pool efficacy of BSA-based gadolinium oxide nanoparticles in large and medium-sized animal models(rabbits)and their effects on the metabolism.In order to prepare a novel type of gadolinium oxide nanoparticles based on human transferrin by simulated biomineralization,the tumor-targeting ability ? T1 performance and biocompatibility were determined by establishing a model of prostate cancer bearing mice.Thus,we expected to explore a new type of MR contrast agents that was more suitable for clinical application,and provide experimental basis for its clinical transformation.Methods: 1.Prepared gadolinium oxide nanoparticles based on bovine serum albumin by simulated biomineralization and characterize such nanoparticles,including their morphology observation with high-definition transmission electron microscopy,particle size analysis,and UV-visible spectroscopy test.The luminometer confirmed the change of the physicochemical structure of protein,and its magnetic resonance imaging performance was known through a magnetic resonance imaging apparatus;and the same method was used to try to prepare and characterize the gadolinium oxide nanoparticle based on human transferrin.2.Definitive blood pool imaging efficacy,toxicity and biocompatibility of bovine serum albumin-based gadolinium oxide nanoparticles(GdNPs-BSA)synthesized in a simulated biomineralization in large and medium animal models(rabbits).4-6 rabbits were randomly divided into a control group and an experimental group.The Gd-DTPA(Magnevist?)and GdNPs-BSA were administered by high-pressure intravenous injection.The imaging of the dorsal aorta,subclavian artery,and renal artery in rabbits was observed.The development of branch vessels in two groups of rabbits was observed.Through the pathological examination to clarify the deposition of two contrast agents in the different tissue,through the dyamic monitoring of blood biochemical indicators to determine its impact on important organs.3.The magnetic resonance imaging ability of Gd@Tf NPs was determined by relaxation rate measurement and magnetic resonance imaging experiments;the in vitro targeting ability of Gd@Tf NPs was determined by cell competition inhibition experiments;TfR-positive prostate cancer cells PC-3 were constructed the mouse model,to determine the efficacy of targeted Gd@Tf NPs for TfR-positive prostate cancer tumors;to record the magnetic resonance signal intensity of each tissue and organ by continuous 2-week MR scanning.ICP-MS were measured to determine possible metabolic pathways of Gd@Tf NPs in mice;in vitro and in vivo toxicity of Gd@Tf NPs was determined by MTT assay and histopathological examination;The two-week changes in hematological parameters and body weights revealed the effects of Gd@Tf NPs on the survival state of mice,thereby exploring the biocompatibility and clearance of Gd@Tf NPs,and looking forward to the possibility of clinical transformation.Results:1.GdNPs-BSA and Gd@Tf NPs were successfully prepared by one-pot method.The high resolution transmission electron microscopy showed that the two kinds of nanoparticles had good dispersibility and small particle size.The particle size of both materials was less than 5nm,which was in accordance with the size requirements of nanomaterials;the specific protein absorption peak of 280 nm was measured by UV-Vis spectrophotometer,indicating that the nano-particles worthy of biomineralization retained the inherent properties of the protein.The traits,ICP-MS and relaxation rate results demonstrate that the gadolinium oxide nanoparticles synthesized by our group have a higher relaxation rate(about 4-5 times)than that of traditional magnesivamide(Magnevist?).2.By injecting equal amounts of Magnevist? and GdNPs-BSA into rabbits,the results showed that GdNPs-BSA had a longer time window for imaging than Magnevist?,which reduced the difficulty of operation.GdNPs-BSA had a better ability to display the dorsal aorta,subclavian artery,and renal artery in rabbits,and could show the branch vessels of the aorta,renal artery,and iliac artery more clarely.The images obtained were clear and could form a clear image with the surrounding tissues.Through pathological observation,no obvious deposition of Gd ions was observed in all major organs of rabbits.Two-week monitoring of blood biochemical parameters showed that the synthesized Gd@Tf NPs did not cause significant damage to important organ functions in rabbits.3.The results of magnetic resonance imaging of cells and mice showed that the nanoparticles had good magnetic resonance imaging performance.Cell competition inhibition assays demonstrated the ability of Gd@Tf NPs to target in vitro.By constructing a TfR-positive prostate cancer mouse model and injecting a small amount of Gd@Tf NPs solution and acquiring magnetic resonance images,it is proved that Gd@Tf NPs had excellent tumor-targeted imaging effects.The in vitro toxicity of Gd@Tf NPs was confirmed by MTT assay.The 2-week mouse pathological examination did not show the deposition of Gd in important organs,indicating its low toxicity in vivo.According to the results of fecal ICP-MS,most of the Gd@Tf NPs were ingested by the liver and then passed into the feces.By observing the hematological parameters and measuring the body weight at 2 weeks,the data of mice after Gd@Tf NPs injection were basically stable,indicating that Gd@Tf NPs had no significant effect on the survival status of mice.Conclusions: 1.Protein-based nanoparticles prepared by biomineralization method have strong operability and reproducibility,and the reaction conditions are mild,and the physicochemical properties of proteins and metal elements are retained to the greatest extent.2.BSA-based gadolinium oxide nanoparticles can successfully perform angiographic imaging in large and medium-sized animals through clinical angiography and obtain good imaging results.GdNPs-BSA not only had good visualization of large vessels and branch vessels,but also has low toxicity and good biocompatibility.It can be used as a clinical magnetic resonance angiography agent to optimize contrast agents.3.Successful synthesis of gadolinium oxide nano-particles based on the Tf,through various in vitro and in vivo experiments showed that the nano-material not only significantly increased the relaxation rate,but also successfully retained the biological function of Tf,can effectively target tumor cells and diseased tissues that express high TfR.At the same time,it was confirmed that the first synthesized Gd@Tf NPs not only had higher stability,lower toxicity,but also can be specifically taken up by the liver and excreted with the feces through the intestines.Therefore,the nanoparticle based on human transferrin prepared by the simulated biomineralization method not only has excellent tumor-specific imaging effect,but also is a new type of low toxicity,high biocompatibility and clearance rate.In summary,the protein-based gadolinium oxide nanoparticles prepared by the simulated biomineralization method is a simple synthesis method with high relaxation rate,high biocompatibility.Also there are significant advantages in MRA imaging and tumor-targeted imaging.