The Study Of Gold Nanodots Enabling Fluorescence Imaging Of Spinal Cord

Purpose:1.Evaluate the effect of fluorescent gold nanoclusters in realizing spinal cord fluorescence imaging.2.Detect the changes in fluorescence imaging after spinal cord injury,and further explore related mechanisms.Materials and Methods:In this study,Ag ND was used as a template to synthesize GSH-coated gold nanocluster reagent AuNDs-GSH through a series of complex chemical processes,and then the physicochemical and optical properties of the fluorescent material were further analyzed.ICR mice were selected as experimental animals for fluorescence imaging of the spinal cord,and the time and concentration effects of AuNDs-GSH on the spinal cord were verified in the non-spinal cord injury group(n=3).The spinal cord injury model was prepared by the Allen method,and the difference between the spinal cord injury site and the non-injured site in fluorescence imaging was compared.In addition,in order to explore the relevant mechanism of AuNDs-GSH imaging the spinal cord,the frozen section of the spinal cord was observed with a laser confocal microscope to understand the distribution of fluorescent particles in the spinal cord tissue.The fluorescence intensity was quantitatively analyzed by Image J.The double overall t test was used to evaluate the difference in the ratio of fluorescence intensity between the spinal cord and the in vitro reagent in the control group and the normal model group.Two-way ANOVA was used to analyze the difference in the ratio of fluorescence intensity between spinal cord tissue and in vitro reagents in different concentration groups.The experimental data are expressed as mean±standard deviation(Mean±SD).Statistical analysis uses Graph Pad Prism.Results:1.AuNDs-GSH is a spherical structure with uniform size and shape,with an average particle size between 2-3nm.The fluorescence intensity emitted by the excitation light at 395-405 nm is the maximum,and the wavelength range of the maximum fluorescence intensity is between 620-630 nm.Compared with the freshly prepared reagents placed under normal conditions for 1 day and 7 days,the fluorescence spectrum has no significant change.Under different PH(4-10)conditions,its fluorescence intensity does not change significantly.In addition,after the UV excitation light is continuously irradiated,the fluorescence intensity is not significantly weakened for at least 1 hour.As the wavelength increases,its absorbance gradually decreases,and its absorbance decreases significantly in the optimal excitation wavelength range of 395-405 nm.2.In vivo experiments at different time points: the control group has almost no fluorescence(only autofluorescence),and it can be seen that the spinal cord has absorbed some fluorescent molecules at 15 min;fluorescence intensity at different time points: the fluorescence intensity of the spinal cord reaches the maximum at 30 min after administration.For at least 2h,the fluorescence intensity of the model group was significantly higher than the autofluorescence of the control group.Compared with the control group,the model group had a statistical difference of P<0.05.The results of in vivo experiments in different concentration groups show that: 1)The spinal cord of the control group has almost no fluorescence(only autofluorescence),and the spinal cord can still show obvious red fluorescence when the concentration is as low as 3mg/ml.2)Fluorescence intensity will increase as the concentration increases.The 4.5mg/ml concentration group is higher than the 3mg/ml group and 4mg/ml group(P<0.05).When the concentration is increased to 5mg/ml,it will be compared with 4.5mg/ml.Compared with the group,there was no significant enhancement(P>0.05).3.After the spinal cord injury model was prepared,obvious irregular bleeding areas were seen at the L1 site;30 minutes after the administration,the fluorescence signal defect or weakening of the spinal cord LI level was seen.Through further observation of the frozen section specimens,it was found that the fluorescence molecules of the undamaged spinal cord tissue were distributed uniformly,and there were scattered local high fluorescence signals.The fluorescence signal of the damaged part is obviously dim,which indicates that the absorption capacity of the damaged part for fluorescent particles is obviously weakened.Conclusion:1.The gold nanocluster reagent AuNDs-GSH prepared in this study has good optical properties and physical and chemical stability.GSH as a ligand makes the structure of AuNDs more stable.2.AuNDs-GSH can make the spinal cord obtain a good fluorescence imaging effect,which is due to the mechanism by which fluorescent particles are absorbed into the spinal cord parenchyma.3.AuNDs-GSH can realize the visual detection of spinal cord injury,and there is obvious fluorescence defect or weakening in the injured part,which means that fluorescence imaging can distinguish the spinal cord injury area by direct vision.