Using RGD-containing Peptide To Enhance Cell Uptake For The Detection Of Furin Activity In Cells

RGD peptide is a short peptide consisting of three amino acids of arginine(Arg)-glycine(Gly)-aspartic acid(Asp).It is widely present in the body,RGD peptide can specificly binding the integrin ?v?3,and mediating a variety of pathophysiological processes.This integrin receptor is highly expressed on the surface of a variety of tumor cells and on the neovascular endothelial cell membrane.The RGD peptide can be used as a specific targeting receptor for highly expressed integrin ?v?3,tumor cells.RGD polypeptide sequence has important biological application,but the linear RGD sequence structure is relatively flexible,unstable and easy to be hydrolyzed by various hydrolases in vivo,leading to the limitation of the application of linear RGD in cells and in vivo.The design of making polypeptide into a ring structure is one of the effective methods for improving the biological stability of the polypeptide.However,the current RGD peptide cyclization methods are mostly biologically incompatible,thus limit their biological applications.Assembly and disassembly are two common and important processes in cells.Due to the aggregation-induced quenching effect,the assembly of the fluorescent dye can lead to the quenching of the fluorescence,and the disassembly can cause the fluorescence signal to reappear.Therefore,the assembly-disassembly of the probe can be used to develop the probe of the fluorescent signal switch.Furin can specifically cleave the polypeptide RRVR sequence and is highly expressed in a variety of tumor cells.There have been some works report of furin-controlled nanoparticle self-assembly and disassembly fluorescence imaging techniques,but since the nanoparticles themselves need to pass through endocytosis into cells,it is more difficult to be absorbed by cells,and the above nanoparticles lack specificly selectivity,resulting in lower detection sensitivity.Therefore,it is very important to develop an assembly-disassembly cell fluorescent probe with targeted effects.In this paper,compound 1,which contains the RGD sequence of the target binding integrin ?v?3,which can be specifically cleaved by the flinar enzyme and the fluorescent chromophore FITC,and the bisulfide bond can be reduced to the mercapto group by the reducing agent,The biodegradable CBT-Cys click reaction,1,2-aminothiol and cyanide condensation,the formation of cyclic RGD containing dimer 1-Dimer,through the self-assembly to form nanoparticles,resulting in FITC fluorescence quenching.The RRVR sequence was cleaved by the nanoparticles after the action with the flumin enzyme,and the FITC fluorescence was recovered to achieve the goal of targeting the tumor cells and imaging them.After the synthesis of the compound and its characterization,the self-assembly characteristics of the compound,the "on-off" response characteristic of the compound,the specific binding to the ?v?3,receptor,and the specificity of the ?v?3,receptor,and the specificly response to furin enzyme were demonstrated by in vitro digestion and cell experiments.