Targeting Collagen By SERS/MRI Peptide Probes And Its Application In The Diagnosis Of Hepatic Fibrosis

Collagen,as an important structural and functional protein in the human body,is a key biomarker for serious diseases such as hepatic fibrosis.The study found that the abnormal deposition of different types of collagens such as type Ⅰ and IV and the degeneration of the characteristic triple helical structure of collagen play a decisive role in the occurrence and development of hepatic fibrosis.Therefore,the development of peptide probes that can simultaneously target and detect multiple collagens is crucial for the diagnosis and treatment of a large number of collagen-related diseases such as hepatic fibrosis.Surface-enhanced Raman spectroscopy（SERS）has the advantages of narrow emission spectrum and multiplexing,and has attracted much attention in the detection of complex biological samples;magnetic resonance imaging（MRI）is the most commonly used clinical imaging tool.This thesis aims to develop novel SERS/MRI peptide probes that can specifically identify different subtypes and structurally denatured collagens,and to explore its application in the detection of hepatic fibrosis and other diseases.The main contents are as follows:1)The specific detection of denatured collagen has always been a huge challenge due to its unique structure.We constructed SERS peptide probes Ag@R1@DCTP and Ag@R2@ICTP that can target and bind to denatured collagen and type Ⅰ collagen.Tissue imaging studies have shown that the simultaneous use of two SERS probes can achieve precise localization of denatured collagen in mouse tail and tendon tissues,eliminating the interference of non-specifically bound SERS signals in tissue imaging.This method has been successfully used for targeted imaging of denatured collagen in tumor pathological tissues such as gastric cancer,lung cancer,intestinal cancer,and esophageal cancer.The multi-probe and co-imaging technology provides a powerful SERS tool for the precise identification of collagen and a potential direction for the specific SERS detection of protein markers in complex biological tissues.2)SERS/fluorescent peptide probes Ag@R1@F-ICTP-DD,Ag@R2@F-IVCTP-DDD and Ag@R3@F-DCTP-D can recognize type Ⅰ,type Ⅳ and denatured collagen with high specificity,respectively.By introducing different amounts of negatively charged amino acid Asp,this series of peptide probes showed good aqueous dispersion and stability.Tissue imaging studies have shown that by fluorescence-guided SERS imaging,we precisely identify changes in the content and distribution of different types of collagens.Collagen fingerprinting in hepatic fibrosis tissue reveals a new mechanism of collagen in the development of hepatic fibrosis.This high-throughput collagen molecular fingerprinting strategy is of great significance for the accurate staging of hepatic fibrosis.This strategy is suitable for the simultaneous detection of multiple markers in complex biological systems,and the establishment of protein fingerprints is of great significance for the diagnosis,treatment and prognosis of various diseases.3)3D printed a microneedle chip containing denatured collagen-targeting peptides to efficiently capture denatured collagen in solution samples.We further utilized the developed SERS peptide probes Ag@R1@IVCTP-DDD and Ag@R2@ICTP-DD to specifically detect the captured denatured type Ⅰ collagen and denatured type Ⅳ collagen,respectively.The microneedle chip showed good specificity for denatured type Ⅰ collagen and denatured type Ⅳ collagen without interference from other common proteins.This method was successfully used to detect collagen in liver extracts and serum of mice with different stages of hepatic fibrosis from S1 to S4.We found that the serum of mice with hepatic fibrosis was mainly type Ⅳ collagen,and its concentration gradually increased from 5.44 ng/m L in S1 to 36.65 ng/m L in S4.The new SERS microneedle chip provides a highly sensitive and specificity detection method for collagen in blood,and has great application potential in the early screening of collagen-related diseases.4)By replacing Pro at the X position with hyp with a special conformation,we designed a peptide probe（Ghyp O）₁₀ with an absolute single-chain conformation,which transformed the polypeptide probe from a stable triple helix structure to a complete single-chain state.The probe does not require any pretreatment,and can recognize denatured collagen in different tissues with high specificity,without binding to normal collagen at all,and can be used for highly sensitive tissue fluorescence imaging in hepatic fibrosis mice.We further constructed the MRI peptide probe Gd-DOTA-Ghyp O of denatured collagen,and successfully used it to image denatured collagen in vivo in mice with different stages of hepatic fibrosis.The novel MRI-targeted peptide probe provides a powerful tool for real-time,highly sensitive,and highly specific detection of denatured collagen in vivo,and is of great significance for the clinical diagnosis of collagen-related diseases such as hepatic fibrosis.