Specific Imaging Of Pathological Collagen

Collagen is the most abundant protein in the human body and a key biomarker for many diseases.As the main component of the extracellular matrix,collagen plays an important role in the basic physiological processes of cell adhesion,proliferation,differentiation and metastasis,and its abnormal remodeling is considered as determinant factors for diseases such as fibrosis,arthritis and cancer.Excessive deposition of type I collagen is often found in fibrosis-related diseases,while exceptional degradation of type II collagen is common in patients with rheumatoid arthritis and osteoarthritis.The anomalous distribution of different types of collagen is closely involved in tumor growth,invasion and metastasis.Therefore,efficient detection of pathological collagen is crucial for the mechanism studies,diagnosis,treatment and prognosis of these diseases.This thesis aims to develop peptide probes for specifically targeting pathological collagen and establish corresponding tissue imaging methods.The main contents are as follows:Chapter I:The specific detection of pathological collagen has always been a huge challenge due to its unique structure.Recent research has found that peptide probes with(Gly-Pro-Hyp)_n repeats in single-stranded state can selectively bind to the partial unfolding sites in pathological collagen.However,such peptide probes spontaneously form trimers,thereby losing their ability to recognize pathological collagen.For the first time,we herein introduced aminoproline into the middle position of the peptide sequence and modified it with the large hindered carboxyfluorescein dye.The novel fluorescent peptide probe maintains stable monomer conformation and can specifically target pathological collagen in various tissues.The peptide probe does not require heat or ultraviolet pretreatment,eliminates the potential risk of tissue damage,and has great potential in the diagnosis and treatment of collagen-related diseases.Chapter II:We have constructed a series of collagen targeting peptide probes PCTP-Dn(n is the number of Asp)by linking the(Gly-Pro-Hyp)₇ sequence with a number of charged amino acids Asp.The stability of the triple helical structure of the peptide probes gradually weakened with the increase in the number of Asp,and the peptide probe PCTP-D6 maintained mainly single-stranded conformation under physiological conditions.The novel type of peptide probes can inhibit the formation of trimers of(Gly-Pro-Hyp)_n sequences by electrostatic repulsion,and they can robustly detect pathological collagen in different types of animal connective tissues and human pathological tissues,which show promising applications in the mechanism studies of collagen-related diseases.Chapter III:We have herein for the first time constructed a new peptide probe F-GOP-10 composed of a repetitive(Gly-Hyp-Pro)_n sequence,which maintains single-stranded conformation even at 0°C.F-GOP-10 can selectively recognize pathological collagen in osteoarthritis tissues,but does not stain normal cartilage tissues at all.F-GOP-10 has been successfully applied to stain liver fibrosis and various cancer tissues,demonstrating that it is a powerful tool for targeting pathological collagen.Notably,peptide probes with different lengths of the Gly-Hyp-Pro triplets all maintain the single stranded conformation,demonstrating their intrinsically nontrimerizing nature.The 100%pure single-stranded peptide probe can be accurately quantified,and can be used for staining paraffin sections and frozen sections,which has great potential in clinical applications.Chapter IV:Quantum dots(QDs)display excellent optical properties such as high brightness and convenient wavelength tuning,and have been widely used in the field of biosensors.However,the conjugation of targeting peptides with quantum dots to construct stable QDs-peptide probes remains very challenging.We have for the first time created a host-guest peptide system to develop QDs-peptide probes with exceptional stability and selectivity.We have discovered that short peptides containing Cys and negatively charged amino acids can be used as host peptides to greatly enhance the stability and water solubility of quantum dots.Meanwhile,guest peptides containing Cys and targeting sequences can be easily bond non-covalently to the quantum dots.Quantum dots modified with different host-guest peptides all exhibit excellent stability and targeting capability,and have been successfully applied for imaging of organelles,membrane proteins and extracellular matrix proteins as well as multi-component imaging of 3D tumor cell models.The host-guest peptide-quantum dot system provides a versatile,simple and robust method for the construction of stable QDs-peptide probes for targeting collagen in cells and tissues.Chapter V:The pathogenesis of collagen leading to fibrosis,cancer and other diseases is a complex process,which involves various types of collagen.By using the newly established host-guest peptide-quantum dot system,we have constructed a variety of QDs-peptide probes with excellent stability and selectivity,which can specifically target type I,II,IV and pathological collagen simultaneously.Due to the large steric effect of quantum dots,the quantum dots-peptide probe targeting pathological collagen always maintains the single-stranded state,completely avoiding the risk of tissue damage caused by heat or ultraviolet pretreatments.In addition,the excellent wavelength-tuning capability of quantum dots endows different peptide probes with distinct colors,which facilitates co-imaging of multiple components.This novel QDs-peptide probe has been successfully applied for imaging different types of collagen involved in fibrosis,arthritis and embryonic development.The QDs-collagen targeting peptide probe system provides an efficient toolbox for simultaneous,highly sensitive,and highly specific detection of various types of collagen,which opens new opportunities for the mechanism studies and early diagnosis of serious diseases such as fibrosis and cancer.