Construction Of Collagen-targeting Peptide Probes And Their Applications In Tissue Imaging

As the principal component of the extracellular matrix,collagen plays an important role in various physiological processes such as cell differentiation and migration,tissue remodeling and organ morphogenesis.As a key structural protein,excessive degradation of collagen leads to severe diseases such as tumors and arthritis.Collagen is an essential component of the tumor microenvironment,and extensive clinical data have revealed that it is a key prognostic factor for cancer differentiation,invasion and metastasis as well as cancer stage.Therefore,the development of robust assays for collagen is crucial for the diagnosis and therapeutics for various collagen-involved diseases such as tumors.This thesis aims to discover potent collagen-targeting peptide probes and explore their tissue imaging applications.The main contents are as follows:Chapter 1:We have given a brief introduction to the sequence pattern,structure and function of collagen,and summarized the research progress of collagen-related diseases,collagen assays,and particularly,collagen-targeting peptide probes.Chapter 2:We have constructed two peptide probes FAM-GOO and FAM-GPP consisting of the repetitive?Gly-Hyp-Hyp?₈ and?Gly-Pro-Pro?₈ sequences,respectively.They both showed superior capability to recognize denatured collagen with high specificity,and FAM-GPP remarkably did not need the preheating treatment.The tissue staining results have shown that both FAM-GOO and FAM-GPP are broad-spectrum probes for selectively detecting denatured collagen of different types and from different species.Importantly,FAM-GPP possessed the unique capability to maintain the monomer conformation by itself,thus avoiding the potential risks of the thermal or UV pretreatment.This novel peptide probe provides a handy and versatile biosensor for specifically targeting denatured collagen,which has attractive potential in the diagnosis and therapeutics of collagen-involved diseases.Chapter 3:We have constructed a series of peptide probes consisting of the?GPO?₃GXO?GPO?₄ and?GPO?₂GXOGPOGXO?GPO?₃ sequences,and systematically investigated the effect of the identity of the residue at the X position on the collagen-targeting capability.We have found two peptide probes FAM-GYO and FAM-GFO with the aromatic residues Tyr and Phe in the X position displayed excellent binding affinity toward pathological collagen.These two peptide probes provided a new perspective to construct collagen-targeting peptides with less triple helical propensity and offered a robust tool for detecting pathological collagen with high specificity and sensitivity.Chapter 4:Cyclic peptides have attractive features such as more rigid structure,higher enzymatic stability and excellent pharmacological properties.We have used the phthalaldehyde?OPA?reaction of lysine and cysteine to achieve the cyclization of the?Gly-Pro-Hyp?_n sequence,and created a cyclic peptide probe FAM-cCTP.The cyclic peptide probe has always maintained the single stranded state,displayed high specificity of pathological collagen,and has been successfully applied for imaging studies of mice and human tissues.The peptide probe synthesized by the cyclization strategy does not need the heating pretreatment,which overcomes the defects of the traditional?Gly-Pro-Hyp?_n probe.It provides a novel tool for specific detection of pathological collagen and effective staining of pathological tissues.