Research On Fluorescence Detection Technology Of Vascular Endothelial Injury-related Factors

Occlusive atherosclerosis and arteriovenous thrombosis are the two main and most common diseases in vascular surgery.Their occurrence and development are closely linked to many cytokines in the body.As the initial link of these two diseases,intracellular dysfunction plays a very important role in the whole course of the disease.Intravascular dysfunction,as the initial link of these two types of diseases,plays a very important role in the entire course of the disease.Vascular endothelial cells,as the largest heterogeneous "organs" in the human body,are the key interface between the regulation of blood and tissue,and therefore have become potential targets for various cellular active substances.Although vascular endothelial cells have strong selfrepairing ability,they continue to be affected by certain vasoactive substances(NO,SO2),reactive oxygen species ROS(H2O2,O2-,Cl O-),and small molecule biothiols(Cys,Hcy),Inflammatory factors(IL-17,VCAM-1),various immune factors,drugs,microorganisms,etc.,the protective effect of endothelial cells on blood vessels weakened or even disappeared,leading to endothelial dysfunction.As the initial link of various vascular diseases,it mainly causes atherosclerosis,arteriovenous thrombosis,hypertension and other vascular diseases through vasoconstriction,leukocyte adhesion and aggregation,platelet aggregation,oxidative stress injury,smooth muscle proliferation and thrombosis.Since the 21 st century,with the advancement of science and technology,genomics and proteomics have developed rapidly.Humans have gradually evolved from the traditional clinical symptoms,signs,and structural images to the series of molecular changes in the occurrence,development,evaluation treatment of diseases and the prognosis.And medical imaging technology has gradually developed from structural imaging and functional imaging to molecular imaging.The molecular impact technology has emerged as the times require.It combines molecular biology with a variety of modern medical imaging technologies,and proposes the application of new imaging methods that to talk to the life process in vivo at the cellular and molecular level,to diagnose and guide the treatment of diseases at the molecular and gene level,so as to achieve the medical goal of individualized precise and visual treatment.Based on this,in order to better promote the early diagnosis and treatment of clinical vascular diseases and improve the prognosis.We have used fluorescence imaging to design and synthesize several small-molecule fluorescent probes for the detection with high selectivity and sensitivity in some important vasoactive molecules(SO2,reactive oxygen species(Cl O-),biothiols(Cys))and horizontal distribution in vivo,and visually analyze the cell in vitro and in vivo for biological performance of the probe,providing a theoretical basis and feasible solutions for guiding the early diagnosis of related clinical vascular diseases and evaluating the efficacy of treatment.This paper mainly includes the following four parts:1.Starting from the indole sulfonate and aromatic aldehydes with different structures,three new fluorescent probes ISBD,ISPD,and ISND that can detect HSO3-in pure water were designed and synthesized.The detection process can be completed within 1 minute and the detection limit is low.They are 9.21×10-9mol/L(9.21 n M)?4.25×10-8 mol/L(42.5 n M)and 8.11×10-8 mol/L(81.1 n M).By comparing the performance of the three probes,the SO2 /HSO3-fluorescent probe ISND with stronger fluorescence intensity(higher fluorescence quantum yield)/ longer emission wavelength and less interference was screened,and its anti-interference ability and Bioimaging detection has been focused on investigation and found that 17 kinds of interfering ions have little effect on the detection of HSO3-.In addition,cytotoxicity and fluorescence experiments also confirmed that the probe ISND has low toxicity,good membrane permeability,and can complete the detection of SO2/HSO3-in the complex biological environment of plasma and cells,which providing a feasible scheme for further real-time imaging in vivo.2.The naphthalimide was used as the fluorophore and ethyl isocyanoacetate was used as the recognition group.A fluorescent probe NAEC was designed and synthesized for endogenous Cl O-detection in biological environment.Through research,it has been found that the probe has the advantages of fast response(<10s),high sensitivity(low detection limit as low as 4.9 n M),large Stokes shift(100 nm),etc.,and it can quickly,sensitively and specifically detect the level of Cl O-in plasma and cells.Competitive interference experiments have confirmed that 22 kinds of interfering substances including various small molecule biothiols and other ROS in the body have little effect on the detection of Cl O-.In addition,the membrane permeability of NAEC is better,and it can quickly penetrate the cell membrane into the cell for specific Cl O-imaging detection.Compared with the previously reported probe ISND,the Stokes shift of the probe NASF is larger,the energy utilization is higher,and the emission wavelength is longer,which realizes the detection of the endogenous substance Cl O-of the cell.This not only helps fluorescent probes become an effective tool for life medicine researchers to study the distribution and mechanism of Cl O-in vivo,but also for early diagnosis of vascular diseases such as atherosclerosis,arteriovenous thrombosis,individualized treatment,and improving prognosis.It also provides important reference at the molecular level in aspects such as postoperative internal environment steady state monitoring.3.Based on the first two parts of the research results: experimental design ideas and advantages and disadvantages of the probe.The good combination of water solubility of indocyanine green and the luminescence of naphthalimide was used to design and synthesize a ratio double detection based on the structure of naphthalimide and indole sulfonate(HSO3-/Cl O-)fluorescent molecular probe NASF,which can simultaneously complete the qualitative and quantitative detection of HSO3-in high sensitivity and high selectivity.It realizes a multi-purpose probe,and based on different emission channels,it will not constitute mutual interference.Compared with the previous two chapters,the probe NASF has the advantages of a larger Stokes shift(Cl O-: 115 nm,HSO3-: 88 nm),a longer emission wavelength(Cl O-:515 nm,HSO3-:548 nm),a better water solubility(DMF/water=1:99,v/v)and other advantages;besides,the probe NASF is a ratio fluorescence probe,which can detect the fluorescence intensity with two different emission wavelengths in order to provide internal self-calibration,reduce interference from the background,minimize the impact of probe concentration,and improve detection accuracy.In vitro cytotoxicity and imaging experiments show that the probe NASF can well perform the detection of endogenous Cl O-in cells,and can perform intracellular imaging without exogenous addition.Meanwhile,it also has a good prospect for biological application in identifying tumor cells.However,when it is further applied to in vivo tumor tissue imaging,the performance is weak,which may be related to the emission wavelength of the probe NASF(?em=515 nm).Studies have shown that fluorescent tissues have different penetrating capabilities at different wavelengths,and biological tissues in the visible region(350-600 nm)has higher light absorption.Most fluorescence signals are absorbed and scattered by animal hair,skin and other tissues,making it difficult to complete high-quality in-vivo fluorescence imaging.When the wavelength is >600 nm,the transmittance of light of various wavelengths will increase significantly in mouse organs.In the near-infrared range of 650-900 nm,the absorption of these wavelengths by hemoglobin,fat,and water remains low level,therefore,the selection of fluorescent probe labels whose excitation and emission are located in the near infrared(650-900 nm)region is more conducive to optical imaging in vivo,especially deep tissue imaging.4.In view of the poor performance of NASF in in-vivo imaging in Chapter III,this research group started with the only near-infrared optical imaging contrast agent,Indocyanine green which is currently approved by the US FDA.A new fluorescent molecular probe Cys was designed and improved based on the chemical structure of indocyanine green(ICG).Which could achieve the specific and sensitive detection of Cys with a detection limit of 14 n M.It also has near-infrared imaging capabilities similar to indocyanine green,with low biological toxicity and good histocompatibility.Its excitation wavelength and emission wavelength were located in the near-infrared I region(excitation: 710 nm,emission: 820 nm).It not only completes the endogenous Cys imaging of cells and Cys concentration measurement in plasma,but also greatly improves the imaging ability in living tissues.Subsequently,we conducted a preliminary study on its distribution and metabolic pathways in mice by intravenous injection.Experiments showed that the Near-infrared fluorescent signal in mice gradually increased within the first 60 minutes after intravenous injection.After 60 min of injection,the fluorescent signal in mice gradually weakened.When injected for 100 minutes,the fluorescence signal gradually disappeared.In addition,in order to further explore the fluorescence accumulation and signal source of the mice,we performed fluorescence imaging tests on isolated organs of mice at different time points,and found that the fluorescent signals in mice mainly came from the liver and small intestine,There is almost no visible fluorescence signal emitted in other organs(Heart,liver,kidney,lung,spleen).The change trend of fluorescence intensity is consistent with that in vivo.Therefore,we speculated that the probe CYNA has a similar metabolic pathway in vivo as ICG,which is mainly metabolized by the liver and enters the intestine in the original form,and then excreted with the intestine as a prototypeis.The in vivo imaging experiment of the probe CYNA not only verifies its usefulness for the whole-body visualization study of Cys in vivo,but also provides a solid theoretical basis for the next stage of our research group to carry out the synthesis of a new lymphatic contrast agent based on indocyanine green.