Identification Of A Novel Peptide By Phage Display For MR Molecular Imaging Of Acute Cerebral Ischemia Tissue

A series of stress response would be triggered in the brain after acute cerebral ischemia, such as acidosis, amino acid excitotoxicity, accumulation of intracellular Ca2+, free radical damage, inflammation and apoptosis, which indicate the pathological change of stroke. Visualization, positioning of these stress response are conducive to individual therapy and predict prognosis. Molecular Imaging enables the visualisation and characterization of the cellular function in living organisms by special molecular probes, which available for monitoring pathological process after cerebral ischemia. However, there were few reports on the specific molecular probes for stroke molecular imaging, limiting the application of molecular imaging technology. Therefore, it is an urgent need to seek molecular probes for stroke molecular imaging. In the present study, in vivo phage display technology was used to identify peptides that specifically bind to cerebral ischemia tissue, and molecular probe was builded based on special peptide. This dissertation included the following three parts:\.In vivo screening of peptides that specifically bind to cerebral ischemia tissue.The C57 transient right middle cerebral artery occlusion (MCAO) models were established by suture method, and in vivo screening was carryed out by Ph.D.-7TM phage display peptide library. After four rounds of screening, the recovered phages at ipsilateral (ischemic) hemisphere were enriched approximately by 10.4-fold as compared to the first round.120 phage clones from the fourth round were randomly picked, then the peptide-coding DNA inserts were sequenced and translated into corresponding peptide sequences, and 17 peptides were acquired at last. The most frequent phage clone displaying HGGVRLY peptide(named HGG, as the phage named HGG-M13) was selected for further experiments.2. Identification of HGG-M13 phage and HGG peptide homing to the stroke tissue.The speciality of HGG-M13 homing to the stroke tissue was characterized by titering of phage and immunofluorescence in HGG-M13 intravenously administered to MCAO mice, and the speciality was gift by HGG peptide displayed on phage, while not the phage. Further, HGG-M13 bound to neurons but not to astrocytes at the ischemic stroke tissue by double immunofluorescent staining experiments.Subsequently, HGG peptide was synthesized and fluorescently labeled with 5-TAMRA, FITC respectively. The results shown that the HGG was observed at ipsilateral hemisphere after fluorescent labled peptides were administered to MCAO mice, and indicated the speciality of HGG peptide homing to the stroke tissue. Moreover, HGG peptide could interact with oxygen and glucose deprivation(OGD) cultured neurons but not the normal neurons in vitro.3. Build of molecular probe based on HGG peptide and molecular imaging.With the activation of EDC/Sulfa-NHS, carboxyl groups that carried on the surface of Mn-Zn ferrite magnetic nanoparticles(MnZnFe2O4) could reacted with HGG peptide, and the MnZnFe2O4-coupled HGG(HGG-MnZnFe2O4) were produced by this method. The HGG-MnZnFe2O4 with a size of 15.17 nm by transmission electron microscope, a hydrodynamic diameter of 53.98 nm, and a high r2 value of 382.45mM-1S-1. The molecular probe was intravenously injected into the MCAO mice and magnetic resonance imaging demonstrated that HGG-MnZnFe2O4 could uptake by the ischemic stroke tissue.In conclusion, the speciality of HGG peptide homing to the stroke tissue was identified in this study. The molecular probe builded on the basis of HGG peptide could uptake by the ischemic stroke tissue. Further studies are needed for stroke molecular imaging based on HGG peptide.