| Background:Necrosis avid compounds are a class of substances that can specifically bind to a large number of targets exposed by necrotic tissues in organisms and then condense in necrotic tissues.By specifically targeting necrotic tissues,necrosis avid compounds have been used in the diagnosis and detection of necrosis-related diseases,such as accurate quantification of myocardial infarction,evaluation of tissue viability,and evaluation of therapeutic effects after tumor treatment.In addition,necrosis avid compounds labeled by radionuclide also show a great potential in the treatment of solid tumors.Among the necrosis avid compounds,hypericin and its radioactive tracers have been widely studied.However,hypericin has poor water solubility,easy self-polymerization,and phototoxicity,which limits its development and clinical transformation to a certain extent.Therefore,finding a necrosis avid compound with good pharmacokinetic distribution and high biological safety has become a new research direction.Evans Blue(EB)is a synthetic diazo dye,which has a long history as a biological dye and diagnostic agent.Because of its albumin binding and fluorescent properties,EB has been widely used in biomedicine,including blood volume measurement in pregnant women and infants.Recent studies have found that multiple staining of EB and Red-Iodized-Oil Solution(RIO)dye can be used to identify infarcted myocardium in risk myocardium,suggesting that EB may have necrotic affinity.In addition,because the structure of EB is easily modifiable and linkable,a large number of tracers based on EB and EB derivatives have been developed for clinical applications.Therefore,exploring the necrotic affinity of EB and its mechanism of targeting necrosis is conducive to the exploration of new applications of EB.The biological safety of EB and its multi-modal imaging indicate that it has a great potential in clinical transformation.Objectives:This doctoral thesis intends to explore the necrotic affinity of EB and radiolabeled EB tracer(131I-EB)at the levels of living body,tissue and cell,and to explain the mechanism of necrosis targeting property.Methods:1.By establishing multiple necrosis-related animal models,including reperfusion partial liver infarction model in rats,rabbit VX2 tumor spontaneous necrosis model and zebrafish muscular necrosis model,the distribution of EB targeting to necrotic tissues was investigated by fluorescence imaging after intravenous injection of EB solution.2.The Iodogen coating method was used to synthesis radiolabeled EB(131I-EB).By using different technologies,such as SPECT imaging,fluorescence imaging,gamma counting,and autoradiography,its distribution and necrosis-targeting characteristics in rat reperfusion partial liver infarction model were explored.3.After inducing tissue and cell necrosis,we incubated samples with EB-serum albumin solution,and then observed the necrotic affinity of EB at the tissue and cell levels by fluorescence imaging.4.After incubating EB solution with necrotic cells,we explored the intracellular localization of EB by confocal microscropy.By spectroscopy experiments,we explored the interaction between EB and DNA or proteins.Using protein electrophoresis and mass spectrometry,we explored the binding of EB to targeted proteins.Results:1.Successfully established multiple necrosis-related animal models,including reperfusion partial liver infarction model in rats,rabbit VX2 tumor spontaneous necrosis model and zebrafish muscular necrosis model.After 24 hours of intravenous injection of EB solution,EB can selectively accumulate in necrotic liver tissues,necrotic tumor tissues,and necrotic muscle fibers,the difference in EB uptake between necrotic and normal tissues was statistically significant.2.131I-EB was successfully prepared by the Iodogen coating method,and the labeling rate was 97%.SPECT imaging showed that radioactive uptake was gradually concentrated in the necrotic liver area.Gamma count,autoradiography,and in vitro fluorescence imaging showed that the radioactive uptake of 131I-EB in necrotic liver tissues was significantly higher than normal liver tissues.3.Tissue and cell incubation of EB-albumin solution showed red fluorescence in necrotic liver tissues,no red fluorescence in normal liver tissues;necrotic cells showed strong red fluorescence,and nearly no red fluorescence in viable cells.4.Confocal microscopy showed that red fluorescence was mainly located in the nucleus of necrotic cells,with the strongest fluorescence in nucleolus.Spectroscopy experiments showed that EB did not bind to DNA,but interacted with proteins.Protein electrophoresis and mass spectrometry analysis showed that EB mainly bound to albumin in necrotic cells and necrotic tissues.Conclusions:1.Multiple necrosis-related animal models,including reperfusion partial liver infarction model in rats,rabbit VX2 tumor spontaneous necrosis model and zebrafish muscular necrosis model,can be used as the basic platform for the research of necrosis avid compounds.2.This study has confirmed the necrotic affinity of EB at the levels of living body,tissue and cell.The mechanism of targeted necrosis is unlikely related to the exposed DNA in necrotic cells,but related to the binding of a large number of exposed proteins.3.The radiolabeled process of 131I-EB by Iodogen coating method is simple and effective,and the radiolabelling rate is high.Studies have shown that 131I-EB has the same necrotic affinity as EB does.As a multimodal necrosis targeting probe,it may have broad prospects in necrosis targeted imaging and tumor necrosis treatment. |
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