Monitoring Effect Of Nitric Oxide Responsive Aggregation Induced Emission Probe On Osteoarthritis

Objective and significance:Primary osteoarthritis（also known as OA）is a common joint disease that often affects middle-aged and elderly people,but there are rarely simple,reliable,and rapid monitoring methods in treating the disease.Previous study has shown that the severity of primary OA positively correlates with the amount of nitric oxide（NO）in joint cavity.The derivative of tetraphenylethene（TPE）TPE-2NH₂ can react with NO,and emit light that positively correlates with the amount of NO,but it has a low water solubility and poor fluorescence.This study modify TPE-2NH₂ with hydrophilic group long poly（ethylene glycol）chain and anti-inflammatory drug diacerein to form NO responsive aggregation induced emission（AIE）probe TPE-2NH₂-（PEG）₂₄-NH-Diacerein.o-phenylenediamine can react with NO to form benzotriazole,and emit fluorescence.The progression of OA can be assessed with fluorescence intensity from ex vivo monitoring.This study may provide a new development direction for early OA monitoring in the clinic.Methods:（1）Evaluating the monitoring effect of TPE-2NH₂ on OA:After completing synthesis of TPE-2NH₂successfully,we assessed the fluorescence intensity and duration after NO react with different concentrations of TPE-2NH₂.After chondrocytes from 3-7 days’C57BL/6J mouse were divided into group Control and IL-1β（pre-treated with 10 ng/m L IL-1β）,TPE-2NH₂was added and fluorescence intensity,NO concentration and cell number were detected at day 2,4 and 6 respectively.Subsequently,monitoring effect of TPE-2NH₂ on OA in vivo was assessed either.（2）Synthesis and identification of NO responsive AIE probe:After synthesized the NO responsive AIE probe successfully,we identified the chemical structure with ¹H Nuclear Magnetic Resonance Spectra（¹H NMR）and Mass Spectrometry（MS）.（3）Assessing the monitoring effect of NO responsive AIE probe on OA in vitro:After chondrocytes from 3-7 days’C57BL/6J mouse were divided into group Control and IL-1β（pre-treated with IL-1β）,NO responsive AIE probe was added at day2,4 and 6 respectively.Fluorescence intensity,NO concentration and cell number were detected,and evaluated with correlation analysis to assess the monitoring effect of the NO responsive AIE probe.（4）Detecting the monitoring effect of NO responsive AIE probe on OA in vivo:After C57BL/6J mouse in group Control and OA were injected with NO responsive AIE probe intra-articularly at week 1,2 and 3,fluorescence intensity,NO concentration and OARSI score were detected,and evaluated with correlation analysis to assess the monitoring effect of the NO responsive AIE probe on OA.Results:（1）Evaluating the monitoring effect of TPE-2NH₂ on OA:After identification of TPE-2NH₂ with ¹H NMR and Liquid Chromatography Mass Spectrometry（LC-MS）,we found that intensity of fluorescence generated by the reaction between TPE-2NH₂ and NO increased with the rise of TPE-2NH₂concentration,and the strongest period sustained from 5 to 10 minutes.In in vitro study,dead cells in group IL-1βincreased from day 2 to 6,and NO concentration and fluorescence intensity rose as well.The monitoring effect of TPE-2NH₂ on OA for in vivo animal experiments is poor.（2）Synthesis and identification of NO responsive AIE probe:The NO responsive AIE probe TPE-2NH₂-（PEG）₂₄-NH-Diacerein was successfully synthesized according to the identification of ¹H NMR and MS.（3）Assessing the monitoring effect of NO responsive AIE probe on OA in vitro:In vitro study demonstrated that the NO responsive AIE probe can smartly monitor OA chondrocytes,and the fluorescence intensity positively correlated with NO concentration.（4）Detecting the monitoring effect of NO responsive AIE probe on OA in vivo:NO responsive AIE probe emitted fluorescence that positively correlates with NO concentration and OARSI score from week 1 to 3,which indicated the fluorescence positively correlates with the progression of OA.Conclusion:In vitro and in vivo experiments demonstrated that NO responsive AIE probe emitted fluorescence that positively correlates with the severity of OA,which is expected to be improved for the clinical monitoring of OA.