The Development Of Type ? Collagen-targeted NIR-? Fluorescence Probe And Applying Of The Probe In Early Diagnosis Of Cartilage Degeneration

Part 1Objective Articular cartilage is a complex functional water-bearing tissue constituting of chondrocytes,proteoglycans,and collagen,and covering the surface of joint,providing smooth contact surfaces and reducing friction and vibration when the joint moving.The degeneration of articular cartilage,occurring in the early stage and developing throughout the whole course of osteoarthritis(OA),is one of the main pathological changes of OA.However,current approaches employed in clinic are incapable to detect the degradation of articular cartilage in the early stage of OA,when change is clinically and radiographically silent.Therefore,the development of new strategies to achieve early diagnosis of articular cartilage degeneration is particularly important for the clinical diagnosis and early intervention of osteoarthritis.Near-infrared(NIR)fluorescence imaging technology has attracted extensive attention and rapid development in medical molecular imaging because of its fast and real-time display,high resolution and high sensitivity,relatively low cost and ease of use,which brings a new dawn to solve the problem of early diagnosis of articular cartilage degeneration.In the first part of this project,a near-infrared second-window fluorescent probe was constructed by using near-infrared second-window(NIR-?)fluorophore CH1055 and type ? collagen targeting peptide and its chemical properties was characterized.Methods The near-infrared second-window molecular probe CH1055-WL was chemically synthesized using the near-infrared second-window fluorophore CH1055 and the type ? collagen targeting peptide WYRGRL.The control probe CH1055-YW was synthesized with CH1055 and the scrambled peptide YRLGRW.The probe molecular weight was then determined by matrix-assisted laser desorption ionization time of flight mass spectrometry(MALDI-TOF-MS).The absorption peak and emission peak were measured under 808 nm laser excitation.The stability of CH1055-WL in different solutes and continuous laser irradiation was evaluated.Results The molecular weights of the probe CH1055-WL and the control probe CH1055-YW were both 1.802 kDa.The absorption peak is at ~750nm and the emission peak is at ~1055nm.CH1055-WL is stable in PBS,medium and serum,and exhibits excellent light stability far superior to indocyanine green(ICG)under continuous laser irradiation.Conclusions The NIR-? fluorescent probe CH1055-WL synthesized by the fluorophore CH1055 and the type ? collagen targeting peptide WYRGRL has the characteristics of small molecular weight,excellent solute stability and photostability.Part 2Objective In vitro assessment of whether CH1055-WL binds to articular cartilage tissue,and binding properties of CH1055-WL to articular cartilage,and detects cytotoxicity of the probe in vitro.Methods The binding properties of the probe CH1055-WL to cartilage were evaluated using fresh pig knee joint cartilage tissue,including probe uptake capacity,probe affinity,and probe retention ability.In vitro cytotoxicity of probe CH1055-WL was determined by MTT assay in human osteoblast cell line hFOB1.19 and rabbit chondrocytes.Results The probe CH1055-WL can bind to articular cartilage tissue to visualize the cartilage in the near-infrared window,and its binding is specific and highly efficient,and can be blocked by the excess peptide WYRGRL.The binding of CH1055-WL to cartilage tissue is saturated within 6-8 hours and has a strong retention capacity within24 hours.Different concentrations of CH1055-WL showed no significant cytotoxicity to human osteoblast cell line hFOB1.19 and rabbit chondrocytes.Conclusions CH1055-WL can efficiently and specifically bind to articular cartilage tissue to image cartilage without showing obvious cytotoxicity.Part 3Objective The ability of CH1055-WL to bind to articular cartilage and binding properties of CH1055-WL to articular cartilage was assessed in normal nude mice.Whether CH1055-WL can detect early articular cartilage degeneration in osteoarthritis model rats was assessed.Methods The in vivo binding of probe to cartilage was evaluated by measuring the fluorescence intensity of the knee joint at different time points after injection of the probe into the knee cavity of normal nude mice.An age-related osteoarthritis model and a surgically induced osteoarthritis model(ACLT+DMM)were constructed using nude mice,and then the effect of detecting the early cartilage degeneration by the change in fluorescence intensity was verified in an arthritis model.Results In normal knee osteoarticular tissues of nude mice,the probe CH1055-WLhas higher retention rate and longer retention time than the control group(CH1055-WL+ blocker).Over time,free probes in the joint cavity are quickly cleared out by the tissue.The fluorescence intensity of the knee joint in the CH1055-WL injection group was significantly higher than that on the control side,indicating that the probe CH1055-WL can specifically target the articular cartilage in nude mice,so that the articular cartilage is imaged in the near-infrared region.Subsequently,we evaluated the ability of probe CH1055-WL to detect early articular cartilage degeneration in nude mice with age-related osteoarthritis model and surgically induced osteoarthritis model.The results showed that the type ? collagen content in the early degenerated knee articular cartilage tissue was lower than that in the normal knee joint,both in the age-related osteoarthritis model and in the surgically induced osteoarthritis model.The results of near-infrared imaging showed that the fluorescence intensity of the injured knee joint decreased faster with time.The fluorescence intensity at 12-24 h after probe injection was significantly lower than that of the normal knee joint.It is indicated that CH1055-WL can truly reflect the change of type ? collagen content in articular cartilage by the intensity of fluorescence intensity,thus realizing the visual detection of early articular cartilage degeneration.Conclusions The probe CH1055-WL can specifically and targetedly bind articular cartilage in vivo to image articular cartilage in the near-infrared region.CH1055-WL can realize the visual diagnosis of early articular cartilage degeneration by detecting the change of type ? collagen content in articular cartilage,which provides a possibility for early intervention of cartilage degeneration and reduction of osteoarthritis.