Construction Of Carbon Quantum Dots Based Nanoprobes And Their Applications In Targeting Detection And Scavenging Of Reactive Oxygen Species

Reactive oxygen species(ROS)are inevitable metabolites of aerobic organisms in the process of metabolism.Under normal physiological conditions,the production and elimination of ROS are in a delicate balance,and the appropriate amount of ROS is important and beneficial to many physiological activities such as immunity and signal transduction.However,due to some exogenous and/or endogenous stimuli,overproduction and accumulation of ROS may happen,which cause oxidative stress and trigger irreversible injury to biomolecules including proteins,DNA and RNA,and further lead to a variety of severe diseases,such as stroke,diabetes,neurodegenerative diseases,and cancer.Therefore,developing analytical methods featuring high sensitivity,high selectivity and rapid response to monitor the production and concentration changes of intracellular ROS,as well as timely and efficiently in situ remove the excessively produced ROS,are of great significance for maintaining normal cellular physiological environment,and preventing and treating ROS-related physiological diseases.On the other hand,carbon quantum dots(CDs)are a new kind of fluorescent nanomateirals.CDs take advantages of simple synthesis,stable optical properties,rich surface chemistry,low cytotoxicity and good biocompatibility,etc.,and have been widely used in biological and medical fields.By means of hetero-atoms doping and surface modification,it is expected to construct a class of novel nanoprobes with good detection and scavenging ability of ROS,and further realize the targeting detection and scavenging of ROS in specific organelles.In this dissertation,a variety of CDs based nanoprobes with targeting detection and scavenging ability of ROS in mitochondria,lysosomes and endoplasmic reticulum were constructed by surface modification and selenium(Se)or tellurium(Te)elements doping approaches.These nanoprobes have potential applications in fields of biology and medicine.The specific research contents and results are as follows:(1)Amino functionalized CDs were first synthesized by a microwave method using1,2,4-triaminobenzene dihydrochloride as the raw material.Ratiometric fluorescent nanoprobe CCA-TPP@CDs with specific detection of hydroxyl radical(·OH)and mitochondrion targeting ability was then prepared by covalent modification of CDs with coumarin-3-carboxylic acid and(4-Carboxybutyl)-triphenylphosphonium bromide(TPP)through amidation reaction.The morphological and chemical composition analysis confirmed the good dispersability,uniform size and successful grafting of functional groups of CCA-TPP@CDs.In vitro experiments showed that that CCA-TPP@CDs could selectively detect·OH with a wide linear detection range(0.1-160?M)and a low detection limit(70 n M).Intracellular tests showed the low cytotoxicity,good biocompatibility,and mitochondrion targeting ability of CCA-TPP@CDs.Based on these merits,CCA-TPP@CDs has been successfully applied in the detection of exogenous or endogenous·OH in mitochondria.(2)Se doped CDs(Se-CDs)were first synthesized by a hydrothermal method using selenocystine as raw material.Then the antioxidant Lyso-Se-CDs was constructed by covalent modification of Se-CDs with morpholine through amidation to targeting removal of·OH in lysosomes.The prepared Lyso-Se-CDs showed uniform size,good dispersibility and colloidal stability.In vitro experiments revealed the good scavenging properties of Lyso-Se-CDs toward·OH due to its good redox property,while intracellular experiments demonstrated the low cytotoxicity,good biocompatibility,lysosome targeting ability,and scavenging ability of excess·OH in lysosomes of Lyso-Se-CDs.Subsequently,a mouse ear inflammation model was established.The fluorescent imaging and staining of tissue sections tests proved that Lyso-Se-CDs could effectively alleviate the ear inflammation in mice.(3)3-((2-((4-Methylphenyl)sulfonamido)ethyl)amino)-3-oxopropanoic acid(MOA)with endoplasmic reticulum(ER)targeting ability was first designed and synthesized.The MOA was then covalently grafted on Se-CDs,which was synthesized from selenocystamine by hydrothermal method,yielding ER targeting antioxidant ER-Se-CDs.The structure of MOA,and the morphology and chemical composition of ER-Se-CDs were characterized by a variety of methods,which confirmed the successful synthesis of MOA and ER-Se-CDs.In vitro and intracellular experiments proved the scavenging ability of ER-Se-CDs for·OH,good biocompatibility,and ER targeting ability.Thus ER-Se-CDs could protect cells from oxidative stress caused by high ROS concentration in the ER region.The test in mouse model of ear inflammation suggested that ER-Se-CDs also had anti-inflammatory effect on ROS-induced inflammation.(4)Te doped CDs(Te-CDs)were synthesized by hydrothermal method using2,2'-ditellanediyldianiline as raw material.The experimental results confirmed the relatively small and uniform size,good dispersibility and colloidal stability of Te-CDs.The excellent redox responsiveness of Te-CDs enabled the rapid(?40 s)and selective detection of superoxide anion(O₂^·-)with a wide linear detection range(0.5-16?M)and a low detection limit(80 n M),while the O₂^·-scavenging test proved the good scavenging ability of Te-CDs for O₂^·-.The Te-CDs were then functionalized by TPP group through amidation reaction.The obtained TPP-Te-CDs possessed functions of O₂^·-detection,O₂^·-scavenging,and mitochondrion targeting ability simultaneously after functionalization.In vitro experiments suggested that the TPP functionalization did not influence the O₂^·-detection and scavenging properties of Te-CDs,while intracellular tests revealed the low cytotoxicity,good biocompatibility,and mitochondrion targeting ability of TPP-Te-CDs.The nanoprobe was successfully applied to detect and remove O₂^·-in mitochondria,demonstrating its ability to protect cells from oxidative stress.