Application Of Recombinant Phage For Artery Thrombosis By Near-infrared Imaging

Objectives:Thrombotic complications are common in clinic and have already become a major concern of human health. Cardiopathy, cerebral stroke and venous thromboembolism are the top 3 leading causes of mortality from cardiovascular diseases in China sharing a common pathogenesis that is thrombotic development. Thrombotic development leading to hemadostenosis would cause ischemia, dysfunction and even necrosis in tissues and organs that are usually considered lethal and irreversible. Notable, pulmonary thrombosis lacking specific symptoms is prone to cause misdiagnosis and results into high mortality. A reliable and quick diagnosis method is of clinical importance. At present, there are multiple methods available for thrombosis detection such as ultrasonic, CT, MRI, DSA, SPECT, et.al., but each method has its own drawbacks restricting their application. An economic, sensitive and real-time diagnosis method has become an emergency. Our primary study demonstrated that the P1 Cm (PATENT NO Z 201110341296. X) peptide derived from CTGF specially bound αⅡbβ3 which was highly expressed in platelets. In this study, we tried to display the P1 Cm peptide on the surface of the M13 phage and applied the phage for near-infrared imaging of thrombus.Part 1 Vector construction and the preparation of the phage recombinantMethods:The M13-P1Cm recombinant was constructed by inserting the P1Cm gene after the M13 foot filament. The successful construction of the recombinant was verified by ELISA, PCR and DNA sequencing. The cytotoxicity of the recombinant phage was tested on normal cells.Results:The results from ELISA, PCR, restriction enzyme digestion and DNA sequencing indicated the M 13-P1Cm recombinant was successfully constructed and the phage could specially bind αⅡbβ3. High purified M13-P1Cm was obtained by PEG8000/NaCl. The obtained phage had no effects on cell proliferation by CCK8 assay.Conclusion:The M13-PlCm recombinant was successfully constructed and the phage could specially bind αⅡbβ3 with limited cytotoxicity.Part 2 M 13-PlCm specifically bind to activated platelets in vitro.Methods:The fluorescent phage probes was prepared by mixing the phages with FITC or Cy5.5. The stability of the phage probes was evaluated after removing the free fluorescent molecules. CCK8 experiment was applied to investigate the cytotoxicity of the prepared phage probes. The phage probes and the platelets were co-incubated and their interaction was studied by Flow cytometry and CLSM.Results:The prepared phage probes were stable within 4 weeks and showed limited effects on normal cells including proliferation and apoptosis. Flow cytometry and CLSM suggested the phage probe bound to platelets through interaction with the β3 subunit of allbβ3. Moreover, the fluorescent intensity was greater than that of the single P1 Cm peptide molecule probe.Conclusion:The results indicated the successful preparation of the phage probes which could specifically bind the αⅡbβ3-subunit of integrin on platelets.Part 3 Application of M13-P1Cm probe for thrombus imaging in vivo.Methods:The nudemice thrombosis model was established by injuring the carotid artery with ferric trichloride. The phage probes were administrated through caudal vein injection and the in vivo near-infrared imaging was carried out with a small animal living imaging system. The animal was then sacrificed and the main organs was collected for in vitro imaging. The carotid artery was collected for HE staining and sections of main organs were prepared for probe hybridization.Results:The artery thrombosis model had been successfully established with ferric trichloride. Cy5.5-M13-P1Cm and CY5.5-P1Cm probes could both accumulate in the thrombosis nidi and the former gave a relative greater fluorescent signal and lasted a longer period. The results of the hybridization experiments using sections were in consistent with the in vivo imaging.Conclusion:The recombinant M13-PlCm was successfully applied to in vivo near-infrared imaging of thrombosis. The signal intensity and the lasting period were superior to that of the single molecule probe of CY5.5-P1Cm. Our results provided a basis for the application of phage as a targeting delivery cargo in clinic.