Synthesis And Evaluation Of Based On Nile Blue-type And TSPO-targeted Near-infrared Fluorescent Molecular Probes For Neuroinflammation

Neuroinflammation is an immune response activated by microglia and astrocytes in the central nervous system throughout the pathogenesis of neurodegenerative diseases.Under normal circumstances,neuroinflammation is beneficial to the nervous system.It can repair damaged nerves,but when the inflammatory response is excessive,the pro-inflammatory factors released will cause damage to the nervous system,forming a vicious circle and accelerating the progress of the disease.Therefore,early diagnosis and visualization of neuroinflammation are particularly important.The molecular imaging of neuroinflammation is mostly labeled by radionuclide.Although these imaging techniques can be well applied to the vivo,these instruments are very expensive,difficult to popularize,and have low spatial resolution and insufficient structural information.Near-infrared fluorescence imaging has gradually become a popular field of optical imaging.It has the characteristics of easy operation,strong tissue penetration,non-invasive real-time monitoring,and low background interference of autofluorescence,and can be well applied to the vivo.At present,there are few reports of fluorescent probes for neuroinflammation,so it is of great significance to develop near-infrared fluorescent molecular probes for detecting neuroinflammation!Fluorescent probes usually consist of a recognition group,a link and a fluorophore.Among the numerous markers of neuroinflammation,Translocator Protein(TSPO)is a widely studied and recognized marker of inflammation.Among the specific ligands of TSPO,PK11195 is called"gold ligand".Subsequent studies found that indoleacetamides and quinazolines have better affinity and specificity for TSPO,so we selected these three ligands as the recognition groups for fluorescent probes.At present,the detection of TSPO through these three ligands mostly uses radionuclide imaging,and only a very small part uses fluorescence imaging,but its large molecular weight,large absorption background,and high non-specific binding seriously limit fluorescence Probe application.At present,there is no report on the near-infrared fluorescent probe targeting TSPO.In this paper,the first TSPO-targeting near-infrared fluorescent probe based on Nile blue with excellent luminous performance was synthesized by using Nile blue as fluorophores,which has great potential in the detection and imaging of neuroinflammation.In this project,three samples of near-infrared fluorescent probes were designed and synthesized with TSPO as the target.According to literature review,cells with high expression of TSPO and cells with low expression of TSPO were combined with three probes,and it was found that the probes had a high signal response to the cells with high expression of TSPO at 100?M,while there was no significant change to the cells with low expression of TSPO.On the one hand,the specific binding of the probe to TSPO was proved,on the other hand,the experimental conditions were explored for subsequent experiments.we used an indirect method to measure the K_D value of the probe to TSPO.The K_D value of the probes ZXH-01 to TSPO was 13.77n M,and the K_D value of the probes ZXH-03 to TSPO was 21.17n M,It shows that this series of probes have a very high affinity with TSPO.All three probes have very low cytotoxicity and have no toxic effects on the cells within a concentration of 5?M.Cell imaging experiments show that the probe has good functional imaging effect at 5?M concentration,and the fluorescent probes were positioned in the same way as the mitochondrial dyes,and then the100?M TSPO specific ligand PK11195 is used to compete with the 5?M fluorescent probe.The fluorescence of the probes was significantly weakened to indicate that the binding site was the same as the binding site of PK11195.More importantly,when the probe is at a concentration of 100?M,its fluorescence intensity and TSPO content expression have a very good linear relationship,which makes it possible to develop a fluorescent probe into a neuroinflammation kit.We used other inflammation indicators,NO kit and fluorescent probe to detect the same batch of cells,and both showed the same trend.We use anti-inflammatory drugs to verify the detection effect of the probe.The preliminary experimental results show that the probe detection result is similar to the NO kit test result,but there are also problems such as small differences in the experimental group,and the experimental conditions need to be further improved.The detection of neuroinflammation is of great benefit to the treatment of diseases.We hope to achieve the diagnosis and visualization of neuroinflammation through the synthesis of some small molecular probes,which provides new ideas for broadening the diagnosis of neuroinflammation.Although the currently synthesized probes have great potential for in vitro evaluation,the situation in vivo is not yet known.Therefore,the development and improvement of fluorescent probes will remain our focus in the future.