Preparation Of Functional Fluorescent Carbon Dots And Their Applications In Cellular Ferroptosis

Ferroptosis,a new type of iron-dependent programmed death different from apoptosis,necrosis,and autophagy,is closely related to ischemia-reperfusion injury,Alzheimer’s disease,cancer and other diseases.The design and construction of novel fluorescent probes with different functions to achieve ferroptosis-related biological imaging and monitoring is one of the hotspots in this field.The process of ferroptosis is accompanied by the accumulation of ferrous iron and the increase of reactive oxygen species,which requires fluorescent probes with strong stability and good fluorescence properties.As a new type of fluorescent nanomaterials,carbon dots(CDs)have the advantages of excellent optical properties,easy functionalization,strong stability,high biocompatibility,simple preparation and abundant precursors.It is of great significance to design and construct CDs with different functions and apply them in ferroptosisrelated biological research.Therefore,taking the ferroptosis microenvironment as the premise and application as the demand orientation,we created three kinds of functional CDs that could visually monitor nucleic acid structure and endoplasmic reticulum polarity during ferroptosis and alleviate oxidative damage caused by ferroptosis.The specific content includes:1.Visual monitoring of nucleic acid structures during cellular ferroptosis by using nucleic acid-responsive CDs with robust anti-interference ability to reactive oxygen speciesDirectly visualizing the dynamic structures of nucleic acids during the ferroptosis of living cells is of great importance.Using m-phenylenediamine and semicarbazide hydrochloride,which are easy to form hydrogen bonds with nucleic acid,as precursors,nuclei-targeting CDs that can specifically respond to nucleic acid is synthesized by solvothermal method.The CDs exhibited a large Stokes shift(75 nm),high fluorescence quantum yield(45.86%)and excellent stability.Importantly,CDs show excellent ROS stability before and after binding to DNA,which can be applied to the cellular environment during ferroptosis.The small-sized spherical structures with an average diameter of 2.84 nm and a surface charge of+27.13 mV enable CDs to enter the nucleus and bind to negatively charged nucleic acids through electrostatic interactions.It is indicated that CDs are capable of visually monitoring the dynamic structure of nucleic acids during ferroptosis.The CDs provide a powerful tool for ferroptosis-related biomedical research.2.Polarity-sensitive CDs for visually monitoring cell viability and endoplasmic reticulum change during ferroptosisThe lipid peroxidation of ferroptosis occurs mainly in the endoplasmic reticulum(ER)and leads to a change in its polarity.To sensitively monitor the polarity change of ER during ferroptosis,endoplasmic reticulum-targeted CDs were synthesized by a onestep solvothermal method using o-phenylenediamine as a precursor.Benefiting from the intramolecular charge-transfer process,the fluorescence of CDs is sensitive to polarity.Meanwhile,CDs have excellent ROS stability,long-wave emission at 605 nm,and are insensitive to various metal cations,amino acids,and pH,which provides favorable conditions for sensitive identification of ER polarity changes during ferroptosis.The experimental results showed that the CDs successfully monitored the increase in the polarity of ER during ferroptosis,and realized the visual monitoring of cell activity based on the change of the fluorescence intensity of ER.This provides an important reference and basis for understanding ferroptosis and other biological processes.3.CDs with radical scavenging activity mitigating oxidative damage caused by ferroptosis.The ferroptosis process is accompanied by increases of intracellular ROS and lipid peroxidation,which lead to cellular oxidative damage.Using morin and cysteine as precursor molecules,a nitrogen and sulfur co-doped CDs with free radical scavenging activity was developed as an antioxidant to alleviate the oxidative damage caused by ferroptosis.The CDs have strong photostability,low cytotoxicity,and good biocompatibility,which are beneficial to biological imaging.The CDs have excellent free radical scavenging activity,and the maximum scavenging efficiency for DPPH reaches 90.4%.Studies have shown that CDs can scavenge the excess free radicals generated during ferroptosis to a certain extent and alleviate the oxidative damage caused by ferroptosis.The CDs have potential as an antioxidant drug for the treatment of ferroptosis-related diseases.